2016

239. Peck, A., Sunden, F., Andrews, L.D., Pande, V.S., Herschlag, D. (2016) JMB In Press. Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase. (PDF File)

See also: Faculty of 1000, June 2016

238. Xue, Y., Gracia, B., Herschlag, D., Russell, R., Al-Hashimi, H.M. (2016) Nat. Commun. In Press. Visualizing Formation of an RNA Folding Intermediate through a Fast Highly Modular Secondary Structure Switch.

237. Shi, X.S., Huang, L., Lilley, D.M.J., Harbury, P.A.B., Herschlag, D. (2016) Nat. Chem. Biol. In Press. The Solution Structural Ensembles of RNA Kink-turn Motifs and Their Protein Complexes. (PDF File)

See also: RNA Conformation - Lightening up invisible states, March 2016

236. Chen, B., Lim, S. Kannan, A., Alford, S.,  Sunden, F., Herschlag, D., Dimov, I., Baer, T., Cochran, J. (2016) Nat. Chem. Biol. 2, 76-81. High-throughput analysis and engineering of antibodies, biosensors, and enzymes in microcapillary arrays. (PDF File)

2015

235. Sengupta, R.N., van Schie, S.N.S., Giambasu, G., Dai, Q., Yesselman, J.D., York, D., Piccirilli, J.A., Herschlag, D. (2015) RNA 22,32-48. An Active Site Rearrangement within the Tetrahymena Group I Ribozyme Releases Non-Productive Interactions and Allows Formation of Catalytic Interactions. (PDF File)

234. Gebala, M., Giambasu, G., Lipfert, J., Bisaria, N., Bonilla, S., Li, G., York, D., Herschlag, D. (2015) J. Am. Chem. Soc. 137, 14705-14715. Cation-Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting Studies. (PDF File)

233. Hogan, G., Brown, P., Hershlag, D. (2015) PLoS Biology 11, e1002307. Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and their mRNA Target. (PDF File)

232. Natarajan, A., Yabukarski, F., Lamba, V., Schwans, J., Sunden, F., Herschlag, D (2015) Science (Technical Comment) 349, 936. “Extreme Electric Fields Power Catalysis In The Active Site of Ketosteroid Isomerase.” (PDF File)

Abstract

231. Shi, X., Herschlag, D., Harbury, P. (2015) Methods in Enzymology: Structures of Large RNA Molecules and Their Complexes 558, 75-97. Quantifying Nucleic Acid Ensembles with X-ray Scattering Interferometry. (PDF File)

230. Sunden, F., Peck, A., Salzman, J., Ressl, S., Herschlag, D. (2015) eLife 4, e06181. Extensive Site-directed Mutagenesis Reveals Interconnected Functional Units in the Alkaline Phosphatase Active Site. (Medline) (PDF File) (Supporting Info)

229. Sigala, P., Ruben, E., Liu, C., Piccoli, P., Hohenstein, E., Martinez, T., Schultz, A., Herschlag D. (2015) J. Am. Chem. Soc. 137, 5730-5740. Determination of Hydrogen Bond Structure in Water Versus Aprotic Environments to Test the Relationship Between Length and Stability. (Medline) (PDF File) (Supporting Info)

228. Herschlag, D. (2015) RNA 21, 527-528. Learning from Ribozymes. (Medline) (PDF File)

227. Bisaria, N., Herschlag, D. (2015) Biochem. Soc. Trans. 43, 172-178. Probing the Kinetic and Thermodynamic Consequences of the Tetraloop/Tetraloop Receptor Monovalent Ion-Binding Site in P4-P6 RNA by smFRET. (Medline) (PDF File)

 

226. Herschlag, D., Allred, B.E., Gowrishankar, S. (2015) Curr. Opin. Struct. Biol. 30, 125-133. From Static to Dynamic: The Need for Structural Ensembles and a Predictive Model of RNA Folding and Function. (Medline)(PDF File)



To understand RNA, it is necessary to move beyond a descriptive categorization towards quantitative predictions of its molecular conformations and functional behavior. An incisive approach to understanding the function and folding of biological RNA systems involves characterizing small, simple components that are largely responsible for the behavior of complex systems including helix-junction-helix elements and tertiary motifs. State-of-the-art methods have permitted unprecedented insight into the conformational ensembles of these elements revealing, for example, that conformations of helix-junction-helix elements are confined to a small region of the ensemble, that this region is highly dependent on the junction's topology, and that the correct alignment of tertiary motifs may be a rare conformation on the overall folding landscape. Further characterization of RNA components and continued development of experimental and computational methods with the goal of quantitatively predicting RNA folding and functional behavior will be critical to understanding biological RNA systems

 

225. Greenfield, M., van de Meent, J-W., Pavlichin, D., Mabuchi, H., Wiggins, C.H., Gonzalez, R.L. Jr., Herschlag, D. (2015) BMC Bioinformatics 16, 1-4. Single-molecule Dataset (SMD): A Generalized Storage Format for Raw and Processed Single-molecule Data. (Medline)



Background Single-molecule techniques have emerged as incisive approaches for addressing a wide range of questions arising in contemporary biological research [ 1-4]. The analysis and interpretation of raw single-molecule data benefits greatly from the ongoing development of sophisticated statistical analysis tools that enable accurate inference at the low signal-to-noise ratios frequently associated with these measurements. While a number of groups have released analysis toolkits as open source software [5-14], it remains difficult to compare analysis for experiments performed in different labs due to a lack of standardization.ResultsHere we propose a standardized single-molecule dataset (SMD) file format. SMD is designed to accommodate a wide variety of computer programming languages, single-molecule techniques, and analysis strategies. To facilitate adoption of this format we have made two existing data analysis packages that are used for single-molecule analysis compatible with this format.ConclusionAdoption of a common, standard data file format for sharing raw single-molecule data and analysis outcomes is a critical step for the emerging and powerful single-molecule field, which will benefit both sophisticated users and non-specialists by allowing standardized, transparent, and reproducible analysis practices.

 

224. Shah, N., Colbert, K.N., Enos, M.D., Herschlag, D., Weis, W.I. (2015) J. Biol. Chem. 4, 2175-2188. Three αSNAP and 10 ATP Molecules are Used in SNARE Complex Disassembly by N- ethylmaleimide Sensitive Factor (NSF). (Medline)(PDF File)



The fusion of intracellular membranes is driven by the formation of a highly stable four-helix bundle of SNARE proteins embedded in the vesicle and target membranes. N-Ethylmaleimide sensitive factor recycles SNAREs after fusion by binding to the SNARE complex through an adaptor protein, αSNAP, and using the energy of ATP hydrolysis to disassemble the complex. Although only a single molecule of αSNAP binds to a soluble form of theSNARE complex, we find that three molecules of αSNAP are used for SNARE complex disassembly. We describe an engineered αSNAP trimer that supports more efficient SNARE complex disassembly than monomeric αSNAP. Using the trimerized αSNAP, we find that N-ethylmaleimide-sensitive factor hydrolyzes 10 ATP molecules on average to disassemble a single SNARE complex.

2014

223. Andrews, L., Zalatan, J., Herschlag, D. (2014) Biochemistry 53, 6811-6819. Probing the Origins of Catalytic Discrimination Between Phosphate and Sulfate Monoester Hydrolysis: Comparative Analysis of Alkaline Phosphatase and Protein Tyrosine Phosphatases. (Medline)(PDF File)(Supporting Info)



Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline Phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ~10^10-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test if positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5×10^6 - 7×10^7, lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalyticpreference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphateand sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar hydrogen bond donors within the active site, dominate in defining this reaction specificity.




222. Gleitsman, K., Herschlag, D. (2014) RNA. 11, 1732-1746. A Kinetic and Thermodynamic Framework for the Azoarcus Group I Ribozyme Reaction. (Medline)(PDF File)(Supporting Info)



Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5'-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5'-exon intermediate in self splicing to remain bound subsequent to 5'-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.

 

221. Natarajan, A., Schwans, J.P., Herschlag, D., (2014) J. Am. Chem. Soc 136, 7643-7654. Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site. (Medline)(PDF File)(Supporting Info)



Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr’s) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results

 

220. Khosla, C., Herschlag, D., Crane, D.E., Walsh, C.T. (2014) Biochemistry 53, 2875-2883. Assembly Line Polyketide Synthases: Mechanistic Insights and Unsolved Problems. (Medline)(PDF File)



Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75-106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain-domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.

 

219. Shi, X.S., Bisaria, N., Benz-Moy, T., Bonilla, S., Pavlichin, D., Herschlag, D. (2014) J. Am. Chem. Soc. 136, 6643-6648. Roles of Long-range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme. (Medline)(PDF File)(Supporting Info)



We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA's topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes.

 

218. Schwans, J.P., Hanoian, P., Lengerich, B., Sunden, F., Gonzalez, A., Tsai, Y., Hammes-Schiffer, S., Herschlag, D. (2014) Biochemistry 53, 2541-2555. Experimental and Computational Mutagenesis to Investigate the Positioning of a General Base within an Enzyme Active Site.(Medline)(PDF File)(Supporting Info)



The positioning of catalytic groups within proteins plays an important role in enzyme catalysis, and here we investigate the positioning of the general base in the enzyme ketosteroid isomerase (KSI). The oxygen atoms of Asp38, the general base in KSI, were previously shown to be involved in anion-aromatic interactions with two neighboring Phe residues. Here we ask whether those interactions are sufficient, within the overall protein architecture, to position Asp38 for catalysis or whether the side chains that pack against Asp38 and/or the residues of the structured loop that is capped by Asp38 are necessary to achieve optimal positioning for catalysis. To test positioning, we mutated each of the aforementioned residues, alone and in combinations, in a background with the native Asp general base and in a D38E mutant background, as Glu at position 38 was previously shown to be mispositioned for general base catalysis. These double-mutant cycles reveal positioning effects as large as 10(3)-fold, indicating that structural features in addition to the overall protein architecture and the Phe residues neighboring the carboxylate oxygen atoms play roles in positioning. X-ray crystallography and molecular dynamics simulations suggest that the functional effects arise from both restricting dynamic fluctuations and disfavoring potential mispositioned states. Whereas it may have been anticipated that multiple interactions would be necessary for optimal general base positioning, the energetic contributions from positioning and the nonadditive nature of these interactions are not revealed by structural inspection and require functional dissection. Recognizing the extent, type, and energetic interconnectivity of interactions that contribute to positioning catalytic groups has implications for enzyme evolution and may help reveal the nature and extent of interactions required to design enzymes that rival those found in biology.

 

217. Shi, X.S., Harbury, P.A.B., Beauchamp, K., Herschlag, D. (2014) Proc. Natl. Acad. Sci. U.S.A. 111, E1473-E1480. From a Structural Average to the Conformational Ensemble of a DNA Bulge. (Medline)(PDF File)(Supporting Info)



Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurine-substituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods.

 

216. Lipfert, J., Doniach, S., Das, R., Herschlag, D. (2014) Annu. Rev. Biochem. 83, 19.1-19.29. Understanding Nucleic Acid-Ion Interactions. (Medline)(PDF File)



Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.

 

215. Giambasu, G., Luchko, T., Herschlag, D., York, D., Case, D. (2014) Biophysical Journal 106, 883-894. Ion Counting from Explicit Solvent Simulations and 3D-RISM. (Medline)(PDF File)(Supporting Info)



The ionic atmosphere around nucleic acids remains only partially understood at atomic-level detail. Ion counting (IC) experiments provide a quantitative measure of the ionic atmosphere around nucleic acids and, as such, are a natural route for testing quantitative theoretical approaches. In this article, we replicate IC experiments involving duplex DNA in NaCl(aq) using molecular dynamics (MD) simulation, the three-dimensional reference interaction site model (3D-RISM), and nonlinear Poisson-Boltzmann (NLPB) calculations and test against recent buffer-equilibration atomic emission spectroscopy measurements. Further, we outline the statistical mechanical basis for interpreting IC experiments and clarify the use of specific concentration scales. Near physiological concentrations, MD simulation and 3D-RISM estimates are close to experimental results, but at higher concentrations (>0.7 M), both methods underestimate the number of condensed cations and overestimate the number of excluded anions. The effect of DNA charge on ion and water atmosphere extends 20-25 Å from its surface, yielding layered density profiles. Overall, ion distributions from 3D-RISMs are relatively close to those from corresponding MD simulations, but with less Na(+) binding in grooves and tighter binding to phosphates. NLPB calculations, on the other hand, systematically underestimate the number of condensed cations at almost all concentrations and yield nearly structureless ion distributions that are qualitatively distinct from those generated by both MD simulation and 3D-RISM. These results suggest that MD simulation and 3D-RISM may be further developed to provide quantitative insight into the characterization of the ion atmosphere around nucleic acids and their effect on structure and stability.

2013

214. Wiersma-Koch, H.I., Sunden, F., Herschlag, D. (2013) Biochemistry 51, 9167-9176. Site-Directed Mutagenesis to Map Interactions that Enhance Cognate and Limit Promiscuous Reaction of an Alkaline Phosphatase Superfamily Phosphodiesterase. (Medline)(PDF File)(Supporting Info)



Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition of side chains that interact with the substrate atoms and substituents that project away from the Zn²⁺ bimetallo core.

 

213. Schwans, J., Sunden, F., Gonzalez, A., Tsai, Y., Herschlag, D. (2013) Biochemistry 52, 7840 7855. Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid Isomerase. (Medline)(PDF File)(Supporting Info)



Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, (13)C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic pKa values and energetic effects.

 

212. Greenfield, M., Herschlag, D. (2013) Methods in Molecular Enzymology 530, 281-297. Fluorescently Labeling Synthetic RNAs. (Medline)(PDF File)



This protocol covers the steps required to incorporate N-hydroxysuccinamide (NHS) functionalized fluorophores into synthetic RNAs containing a residue derivatized with a primary amine. This method has been widely used to label RNA oligonucleotides that are used directly, targeted to a complementary RNA using base pairing rules, or covalently ligated to a RNA of interest (Ha et al., 1999; Hodak et al., 2005; Baum and Silverman, 2007; Sattint et al., 2008; Akiyama and Stone, 2009; Solomatin and Herschlag, 2009). While this technique is quite general, the details of a particular experiment can vary, therefore, it is always important to keep in mind that other labeling strategies are available and should potentially be considered.

 

211. Porecha, R., Herschlag, D. (2013) Methods in Molecular Enzymology 530, 253-279. RNA Radiolabeling. (Medline)(PDF File)



Radioactive end-labeling is useful for visualizing and allowing the detection of nucleic acids at trace concentrations. Radioactive end-labeling can be carried out on RNA, DNA, or other modified nucleic acids. For RNA, the uses of end-labeling extend beyond simple detection of the intact RNA. A number of RNA molecules studied by biologists form three-dimensional structures in solution, and many of the techniques used to study these structures depend on the ability to visualize the RNA after fragmentation. Labeling at either the 5'- or 3'-end serves as a gateway into these structural analysis techniques (see Structural Analysis of RNA Backbone Using In-Line Probing), and protocols for these labeling procedures are described below (for a nonradiactive labeling protocol, see Fluorescently Labeling Synthetic RNAs).

 

210. Chen, J., Du Bois, J., Glenn, J., Herschlag, D., Khosla, C. (2013) ACS Chemical Biology 8, 1860-1861. The Stanford Institute for Chemical Biology. (Medline)(PDF File)

 

209. Nguyen, P., Shi, X., Sigurdson, S., Herschlag, D., Qin, P. (2013) ChemBioChem 14, 1720-1723. A Single-stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme. (Medline)(PDF File)(Supporting Info)



Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes.

 

208. Schwans, J.P., Sunden, F., Lassila, J.K., Gonzalez, A., Tsai, Y., Herschlag, D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 11308-11313. Use of Anion-Aromatic Interactions to Position the General Base in the Ketosteroid Isomerase Active Site. Faculty of 1000 Biology, July 2013 http://f1000.com/prime/718095449 (Medline)(PDF File)(Supporting Info)



Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogen-bonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.

 

207. Sigala, P.A., Fafarman, A.T., Schwans, J.P., Fried, S.D., Fenn, T.D., Caaveiro, J.M.M., Pybus, B., Ringe, D., Petsko, G., Boxer, S., Herschlag, D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E2552-E2561. Quantitative Dissection of Hydrogen Bond-mediated Proton Transfer in the Ketosteroid Isomerase Active Site. (Medline)(PDF File)(Supporting Info)



Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

 

206. Andrews, L., Fenn, T., Herschlag, D. (2013) PLoS Biology 11, 1-18. Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer Enzymes. Faculty of 1000 Biology, July 2013 http://f1000.com/prime/718031351?subscriptioncode=8db293aa-f64a-4bfe-92ae-f6c9dda7cfc3 (Medline)(PDF File) (Supporting Info)



Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the ³¹P chemical shift of bound P(i). The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥10⁸-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P(i) binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.

 

205. Shi, X., Herschlag, D., Harbury, P. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E1444-E1451. The Structural Ensemble and Microscopic Elasticity of Freely Diffusing DNA by Direct Measurement of Fluctuations. (Medline)(PDF File)(Supporting Info)



Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA–protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.

 

204. Herschlag, D., Natarajan, A. (2013) Biochemistry 52, 2050-2067. Fundamental Challenges in Mechanistic Enzymology: Progress toward understanding the Rate Enhancements of Enzymes. (Medline)(PDF File)



Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.

 

203. Ruben, E.A., Schwans, J.P., Gonzalez, A., Tsai, Y., Herschlag, D. (2013) Biochemistry 52, 1074-1081. Ground State Destabilization From a Positioned General Base in the Ketosteroid Isomerase Active Site. (Medline)(PDF File)(Supporting Info)



We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction's Michaelis complex. The analogue binding affinity increased ~1 order of magnitude for the Asp38Asn mutation and ~2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Aspgeneral base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids.

 

2012

202. Sim, A.Y.L., Lipfert, J., Herschlag, D., Doniach, S. (2012) Phys. Rev. E 86, 021901. Salt Dependence of the Radius of Gyration and Flexibility of Single-Stranded DNA in Solution Probed by Small-Angle X-ray Scattering. (Medline)(PDF File)



Short single-stranded nucleic acids are ubiquitous in biological processes; understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small-angle x-ray scattering to study 8–100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T’s structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

 

201. Forconi, M., Benz-Moy, T., Rule Gleitsman, K., Ruben, E., Metz, C. and Herschlag, D. (2012) RNA 18, 1222-1229. Exploring Purine N7 Interactions via Atomic Mutagenesis: The Group I Ribozyme as a Case Study. (Medline)(PDF File)



Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure–function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

 

200. Althoff, E.A., Wang, L., Jiang, L., Giger, L., Lassila, J.K., Wang, Z., Smith, M., Hari, S., Kast, P., Herschlag, D., Hilvert, D., Baker, D. (2012) Protein Science 5, 717-726. Robust Design and Optimization of Retroaldol Enzymes. (Medline) (PDF File) (Supporting Info)



Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water-mediated stabilization of the carbinolamine hydroxyl and b-hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site-directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride-reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited. Keywords: computational protein design; computational enzyme design; enzyme engineering; directed evolution; enzyme; aldolase; rational design

 

 

199. Anthony, P.C., Sim, A.Y.L., Chu, V.B., Doniach, S., Block, S.M. and Herschlag, D. (2012) J. Am. Chem. Soc. 134, 4607-4614. Electrostatics of Nucleic Acid Folding Under Conformational Constraint. (Medline) (PDF File) (Supporting Info)



RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg2+ stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson−Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg2+ titrations. This finding is consistent with previous work, suggesting that Poisson−Boltzmann and other mean-field theories fail for higher valency cations where ion−ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.

 

 

198. Frederiksen, J.K., Li, N-S., Das, R., Herschlag, D. and Piccirilli, J.A. (2012) RNA 18, 1123-1141. Metal Ion Rescue Revisited: Biochemical Detection of Site-bound Metal Ions Important for RNA Folding. (Medline) (PDF File) (Supporting Info)



Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4–P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

 

 

197. Shi, X., Solomatin, S. and Herschlag, D. (2012) J. Am. Chem. Soc. 134, 1910-1913. A Role for a Single-stranded Junction in RNA Catalysis by the Tetrahymena Group I Ribozyme.(Medline)(PDF File)(Supporting Info)



We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme’s catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5′-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymenaintron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

 

 

196. Greenfeld, M., Pavlichin, D.S., Mabuchi, H. and Herschlag, D. (2012) PLoS One 7, e30024 Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data. (Medline) (PDF File)(Supporting Info)



Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART:Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information fromsingle molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in theanalysis of reported data.

 

195. Fafarman, A.T., Sigala, P.A., Schwans, J.P., Fenn, T.D., Herschlag D. and Boxer, S.G. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E299-E308. Quantitative, Directional Measurement of Electric Field Heterogeneity in the Active Site of Ketosteroid Isomerase. (Medline) (PDF File) (Supporting Info)



Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV/cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK(a) prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK(a) near 7 was included. UV-absorbance and (13)C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis.

 

2011

194. Schwans, J., Sunden, F., Gonzalez, A., Tsai, Y. and Herschlag, D. (2011) J. Am. Chem. Soc. 133, 20052-20055. Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase. (Medline)(PDF File)(Supporting Info)



Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be ~650 Å(3) and sufficient for occupancy by 15-17 water molecules and led to a rate decrease of only ~10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.

See also: Correction to "Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid," June 2016

193. Sengupta, R.N., Yoshida, A., Herschlag, D. and Piccirilli, J.A. (2011) ACS Chem. Biol. 7, 294-299. Thermodynamic Evidence for Negative Charge Stabilization by a Catalytic Metal Ion within an RNA Active Site. (Medline)(PDF File)(Supporting Info)



Protein and RNA enzymes that catalyze phosphoryl transfer reactions frequently contain active site metal ions that interact with the nucleophile and leaving group. Mechanistic models generally hinge upon the assumption that the metal ions stabilize negative charge buildup along the reaction coordinate. However, experimental data that test this assumption directly remain difficult to acquire. We have used an RNA substrate bearing a 3'-thiol group to investigate the energetics of a metal ion interaction directly relevant to transition state stabilization in the Tetrahymena group I ribozyme reaction. Our results show that this interaction lowers the pK(a) of the 3'-thiol by 2.6 units, stabilizing the bound 3'-thiolate by 3.6 kcal/mol. These data, combined with prior studies, provide strong evidence that this metal ion interaction facilitates the forward reaction by stabilization of negative charge buildup on the leaving group 3'-oxygen and facilitates the reverse reaction by deprotonation and activation of the nucleophilic 3'-hydroxyl group.

 

192. Bobyr, E., Lassila, J.K., Wiersma-Koch, H.I., Fenn, T.D., Lee, J.J., Nikolic-Hughes, I., Hodgson, K.O., Rees, D.C., Hedman, B. and Herschlag, D. (2011) J. Mol. Biol. 415, 102-117. High-resolution Analysis of Zn²⁺ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography. (Medline)(PDF File)(Supporting Info)



Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn(2+) catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn(2+) coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray crystallographic data. We report a 1.29-Å crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and NPP in the free-enzyme forms, with AMP and inorganic phosphate ground-state analogs and with vanadate transition-state analogs. These studies yielded average zinc-ligand distances in AP and NPP free-enzyme forms and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal-ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the results suggest that the binuclear Zn(2+) catalytic site remains very similar between AP and NPP during the course of a reaction cycle.

 

191. Forconi, M., Schwans, J.P., Porecha, R.H., Sengupta, R.N., Piccirilli, J.A. and Herschlag, D. (2011) Chemistry and Biology 18, 949-954. 2´-Fluoro Substituents Can Mimic Native 2´-Hydroxyls within Structured RNA. (Medline)(PDF File)(Supporting Info)



The ability of fluorine in a C-F bond to act as a hydrogen bond acceptor is controversial. To test such ability in complex RNA macromolecules, we have replaced native 2′-OH groups with 2′-F and 2′-H groups in two related systems, the Tetrahymena group I ribozyme and the ΔC209 P4-P6 RNA domain. In three cases the introduced 2′-F mimics the native 2′-OH group, suggesting that the fluorine atom can accept a hydrogen bond. In each of these cases the native hydroxyl group interacts with a purine exocyclic amine. Our results give insight about the properties of organofluorine and suggest a possible general biochemical signature for tertiary interactions between 2′-hydroxyl groups and exocyclic amino groups within RNA.

 

190. Benz-Moy, T. L. and Herschlag, D. (2011) Biochemistry 50, 8733-8755. Structure-function Analysis from the Outside In: Long-range Tertiary Contacts in RNA Exhibit Distinct Catalytic Roles. (Medline)(PDF File)(Supporting Info)



The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.

 

189. Andrews, L., Deng, H. and Herschlag, D. (2011) J. Am. Chem. Soc. 133, 11621-11631. Isotope-Edited FTIR of Alkaline Phosphatase Resolves Paradoxical Ligand Binding Properties and Suggests a Role for Ground-State Destabilization. (Medline)(PDF File)(Supporting Info)



Escherichia coli alkaline phosphatase (AP) can hydrolyze a variety of chemically diverse phosphate monoesters while making contacts solely to the transferred phosphoryl group and its incoming and outgoing atoms. Strong interactions between AP and the transferred phosphoryl group are not present in the ground state despite the apparent similarity of the phosphoryl group in the ground and transition states. Such modest ground-state affinity is required to curtail substrate saturation and product inhibition and to allow efficient catalysis. To investigate how AP achieves limited affinity for its ground state, we first compared binding affinities of several related AP ligands. This comparison revealed a paradox: AP has a much stronger affinity for inorganic phosphate (P(i)) than for related compounds that are similar to P(i) geometrically and in overall charge but lack a transferable proton. We postulated that the P(i) proton could play an important role via transfer to the nearby anion, the active site serine nucleophile (Ser102), resulting in the attenuation of electrostatic repulsion between bound P(i) and the Ser102 oxyanion and the binding of P(i) in its trianionic form adjacent to a now neutral Ser residue. To test this model, isotope-edited Fourier transform infrared (FTIR) spectroscopy was used to investigate the ionic structure of AP-bound P(i). The FTIR results indicate that the P(i) trianion is bound and, in conjunction with previous studies of pH-dependent P(i) binding and other results, suggest that P(i) dianion transfers its proton to the Ser102 anion of AP. This internal proton-transfer results in stronger P(i) binding presumably because the additional negative charge on the trianionic P(i) allows stronger electrostatic interactions within the AP active site and because the electrostatic repulsion between bound P(i) and anionic Ser102 is eliminated when the transferred P(i) proton neutralizes Ser102. Indeed, when Ser102 is neutralized the P(i) trianion binds AP with a calculated K(d) of ≤290 fM. These results suggest that electrostatic repulsion between Ser102 and negatively charged phosphate ester substrates contributes to catalysis by the preferential destabilization of the reaction's E·S ground state.

 

188. Greenfeld, M., Solomatin, S.V. and Herschlag, D. (2011) J. Biol. Chem. 286, 19872-19879. Removal of Covalent Heterogeneity Reveals Simple Folding Behavior for P4-P6 RNA. (Medline)(PDF File)(Supporting Info)



RNA folding landscapes have been described alternately as simple and as complex. The limited diversity of RNA residues and the ability of RNA to form stable secondary structures prior to adoption of a tertiary structure would appear to simplify folding relative to proteins. Nevertheless, there is considerable evidence for long-lived misfolded RNA states, and these observations have suggested rugged energy landscapes. Recently, single molecule fluorescence resonance energy transfer (smFRET) studies have exposed heterogeneity in many RNAs, consistent with deeply furrowed rugged landscapes. We turned to an RNA of intermediate complexity, the P4-P6 domain from the Tetrahymena group I intron, to address basic questions in RNA folding. P4-P6 exhibited long-lived heterogeneity in smFRET experiments, but the inability to observe exchange in the behavior of individual molecules led us to probe whether there was a non-conformational origin to this heterogeneity. We determined that routine protocols in RNA preparation and purification, including UV shadowing and heat annealing, cause covalent modifications that alter folding behavior. By taking measures to avoid these treatments and by purifying away damaged P4-P6 molecules, we obtained a population of P4-P6 that gave near-uniform behavior in single molecule studies. Thus, the folding landscape of P4-P6 lacks multiple deep furrows that would trap different P4-P6 molecules in different conformations and contrasts with the molecular heterogeneity that has been seen in many smFRET studies of structured RNAs. The simplicity of P4-P6 allowed us to reliably determine the thermodynamic and kinetic effects of metal ions on folding and to now begin to build more detailed models for RNA folding behavior.

 

187. Solomatin, S.V., Greenfeld, M. and Herschlag, D. (2011) Nat. Struct. Mol. Biol. 18, 732–734. Implications of Molecular Heterogeneity for the Cooperativity of Biological Macromolecules. (Medline)(PDF File)(Supporting Info)



Cooperativity, a universal property of biological macromolecules, is typically characterized by a Hill slope, which can provide fundamental information about binding sites and interactions. We demonstrate, through simulations and single-molecule FRET (smFRET) experiments, that molecular heterogeneity lowers bulk cooperativity from the intrinsic value for the individual molecules. As heterogeneity is common in smFRET experiments, appreciation of its influence on fundamental measures of cooperativity is critical for deriving accurate molecular models.

 

186. Forconi, M., Porecha, R. H., Piccirilli, J. A. and Herschlag, D. (2011) J. Am. Chem. Soc. 133, 7791-7800. Tightening of Active Site Interactions En-route to the Transition State Revealed by Single-Atom Substitution in the Guanosine-Binding Site of the Tetrahymena Group I Ribozyme. (Medline)(PDF File)(Supporting Info)



Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.

 

185. Lassila, J.K., Zalatan, J.G. and Herschlag, D. (2011) Annu. Rev. Biochem. 80, 669-702. Biological Phosphoryl Transfer Reactions: Understanding Mechanism and Catalysis. (Medline)(PDF File)(Supporting Info)



Phosphoryl-transfer reactions are central to biology. These reactions also have some of the slowest nonenzymatic rates and thus require enormous rate accelerations from biological catalysts. Despite the central importance of phosphoryl transfer and the fascinating catalytic challenges it presents, substantial confusion persists about the properties of these reactions. This confusion exists despite decades of research on the chemical mechanisms underlying these reactions. Here we review phosphoryl-transfer reactions with the goal of providing the reader with the conceptual and experimental background to understand this body of work, to evaluate new results and proposals, and to apply this understanding to enzymes. We describe likely resolutions to some controversies, while emphasizing the limits of our current approaches and understanding. We apply this understanding to enzyme-catalyzed phosphoryl transfer and provide illustrative examples of how this mechanistic background can guide and deepen our understanding of enzymes and their mechanisms of action. Finally, we present important future challenges for this field.

2010

184. Hanoian, P., Sigala, P.A., Herschlag, D. and Hammes-Schiffer, S. (2010) Biochemistry 49, 10339-10348. Hydrogen Bonding in the Active Site of Ketosteroid Isomerase: Electronic Inductive Effects and Hydrogen Bond Coupling. (Medline)(PDF File)(Supporting Info)

 

183. Riordan, D., Herschlag, D. and Brown, P.O. (2010) Nucl. Acids Res. 39, 1501-1509. Identification of RNA Recognition Elements in the S. cerevisiae Transcriptome. (Medline) (PDF File)

182. Fafarman, A.T., Sigala, P.A., Herschlag, D. and Boxer, S.G. (2010) J. Am. Chem. Soc. 132, 12811-12813. Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen Bonding Effects. (Medline) (PDF File)

181. Porecha, R. and Herschlag, D. (2010) Methods Navigator: Cookbook for Biomedical Labs "RNA Radiolabeling" (Methods Navigator)

180. Greenfeld, M. and Herschlag, D. (2010) Methods Navigator: Cookbook for Biomedical Labs "Fluorescently Labeling Synthetic RNAs." (Methods Navigator)

179. Wan, Y., Suh, H., Russell, R. and Herschlag, D. (2010) J. Mol. Biol. Multiple Unfolding Events During Native Folding of the Tetrahymena Group I Ribozyme. (Medline) (PDF File)

178. Forconi, M., Sengupta, R., Piccirilli, J. A. and Herschlag, D. (2010) Biochemistry 49, 2753-2762. A Rearrangement of the Guanosine-binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site. (Medline) (PDF File)

177. Lassila, J.K., Baker, D. and Herschlag, D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 4937-4942. Origins of Catalysis by Computationally Designed Retroaldolase Enzymes. (Medline) (PDF File)
See also: Faculty of 1000 Biology, http://f1000biology.com/article/id/2953961

176. Kraut, D.A., Sigala, P.A., Fenn, T.D. and Herschlag D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1960-1965 . Dissecting the Paradoxical Effects of Hydrogen Bond Mutations in the Ketosteroid Isomerase Oxyanion Hole. (Medline) (PDF File)

175. Lipfert, J., Sim, A.Y.L., Herschlag, D. and Doniach, S. (2010) RNA 16, 708-719. Dissecting Electrostatic Screening, Specific Ion Binding, and Ligand Binding in an Energetic Model for Glycine Riboswitch Folding. (Medline) (PDF File)

174. Solomatin, S.V., Greenfeld, M., Chu, S. and Herschlag. D. (2010) Nature 463, 681-684. Multiple Native States of an RNA Enzyme Reveal Persistent Ruggedness of an RNA Folding Landscape. (Medline) (PDF File)

173. Ali, M., Lipfert, J., Seifert, S., Herschlag, D. and Doniach, S. (2010) J. Mol. Biol. 396, 153-165. The Ligand-Free State of the TPP Riboswitch, A Partially Folded RNA Structure. (Medline) (PDF File)

172. Greenfeld, M. and Herschlag, D. (2010) Methods in Enzymology 472, 205-20. Measuring the Energetic Coupling of Tertiary Contacts in RNA Folding Using Single Molecule Fluorescence Resonance Energy Transfer. (Medline) (PDF File)

2009

171. Greenfeld, M. and Herschlag, D. (2009) Methods in Enzymology 469, 375-389. Probing Nucleic Acid Ion-Interactions with Buffer Exchange Atomic Emission Spectroscopy. (PDF File)

170. Shi, X and Herschlag, (2009) Methods in Enzymology 469, 287-302. Fluorescence Polarization Anisotropy to Measure RNA Dynamics. (PDF File)

169. Solomatin, S. and Herschlag, D. (2009) Methods in Enzymology 469, 47-67. Methods of Site-Specific Labeling of RNA with Fluorescent Dyes. (PDF File)

168. Forconi M. and Herschlag, D. (2009) Methods in Enzymology 468, 311-333. Use of Phosphorothioates to Identify Sites of Metal Ion Binding in RNA. (PDF File)

167. Forconi, M. and Herschlag, D. (2009) Methods in Enzymology 468, 91-106 Metal Ion-Based RNA Cleavage as a Structural Probe. (PDF File)

166. Chu, V.B., Lipfert, J., Bai, Y., Pande, V.S., Doniach, S., and Herschlag, D. (2009) RNA 15, 2195-2205. Do Conformational Biases of Simple Helical Junctions Influence RNA Folding Stability and Specificity? PMC2779674 (Medline) (PDF File)

165. Hendrickson, D.G., Hogan, D. J., McCullough, H.L., Myers, J.W., Herschlag, D., Ferrell, J.E. and Brown, P.O. (2009) PLoS Biology 7, e1000238. Concordant Regulation of Translation and mRNA Decay for Hundreds of Targets of a Human microRNA. PMC2766070 (Medline) (PDF File)

164. Forconi, M., Sengupta, R.N., Liu, M-C., Sartorelli, A.C., Piccirilli, J. A. and Herschlag, D. (2009) Angew. Chem. Int. Ed.  48, 7171-7175. Structure and Function Converge to Identify a Hydrogen Bond in the Group I Ribozyme Active Site. PMCID in process (Medline) (PDF File)
Selected as ‘Hot Paper’ by the Editors and highlighted in Nat. Chem. Biol. (2009) 5, 712.

163. Schwans, J.P., Kraut, D.A. and Herschlag, D. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 14271-14275. Determining the Catalytic Role of Remote Substrate Binding Interactions in Ketosteroid Isomerase. PMC2732871 (Medline) (PDF File)

162. Zalatan, J.G. and Herschlag, D. (2009) Nature Chem. Biol. 5, 516-520. The Far Reaches of Enzymology. (Medline) (PDF File)

161. Shi, X., Mollova, E., Pljevaljcic, G., Millar, D. and Herschlag, D. (2009) J. Am. Chem. Soc. 131, 9571-9578. Probing the Dynamics of the P1 Helix within the Tetrahymena Group I Intron. PMC2758093 (Medline) (PDF File)

160. Sigala, P.A., Caaveiro, J. M. M., Ringe, D., Petsko, G. A. and Herschlag D. (2009) Biochemistry 48, 6932–6939. Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site. (Medline) (PDF File)

159. Sigala, P.A., Tsuchida, M. A., and Herschlag, D. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 9232-9237 Hydrogen Bond Dynamics in the Active Site of Photoactive Yellow Protein. PMC2695108 (Medline) (PDF File) Biopolymers Research Highlights Volume 91 / Number 9 iii

158. Kraut, D. A., Churchill, M.J., Dawson, P.E. and Herschlag, D. (2009) ACS Chem. Biol. 4, 269-273. Evaluating the Potential for Halogen Bonding in the Oxyanion Hole of Ketosteroid Isomerase Using Unnatural Amino Acid Mutagenesis. PMC2708088 (Medline) (PDF File)

157. Grant, G.P.G., Boyd, N., Herschlag, D. and Qin, P.Z. (2009) J. Am. Chem. Soc. 131, 3136–3137. Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin-Labeling. PMC2788000 (Medline) (PDF File) See also: Faculty of 1000 Biology, Feb 2009 http://www.f1000biology.com/article/id/1156905/evaluation

156. Lipfert, J., Herschlag, D. and Doniach, S. (2009) Methods in Molecular Biology 540, 141-159. Riboswitch Conformations Revealed by Small-Angle X-Ray Scattering.PMCID in process (Medline) (PDF File)

155. Jonikas, M.A., Radmer, R.J., Laederach, A., Das, R., Pearlman, S., Herschlag, D. and
Altman, R.B. (2009) RNA 15, 189-199. Coarse-grained Modeling of Large RNA Molecules with Knowledge-based Potentials and Structural Filters. PMC2648710 (Medline) (PDF File)

2008

154. Chu, V.B., Bai, Y., Lipfert, Y., Doniach, S. and Herschlag, D. (2008) Curr. Opin. Chem. Biol. 12, 619-625. A Repulsive Field: Advances in the Electrostatics of the Ion Atmosphere. (Medline) (PDF File)

153. Lassila, J. and Herschlag, D. (2008) Biochemistry 47, 12853-12859. Promiscuous Sulfatase Activity and Thio-effects in a Phosphodiesterase of the Alkaline Phosphatase Superfamily. (Medline) (PDF File)

152. Zalatan, J., Fenn, T. D., Herschlag, D. (2008) J. Mol. Biol. 384, 1174-1189. Comparative Enzymology in the Alkaline Phosphatase Superfamily to Determine the Catalytic Role of an Active-Site Metal Ion. (Medline) (PDF File)

151. Hogan, D., Riordan, D., Gerber, A., Herschlag, D. and Brown, P. (2008) PloS Biology 6, 2297-2313. Diverse RNA-Binding Proteins Interact with Functionally Related Sets of RNAs, Suggesting an Extensive Regulatory System. (Medline) (PDF File)

150. Sigala, P., Kraut, D., Caaveiro, J., Pybus, B., Ruben, E., Ringe, D., Petsko, G., Herschlag, D., (2008) J. Am. Chem. Soc. 130, 13696-13708. Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole. (Medline) (PDF File)
See also: Nature 2008, 456, 45-47. http://www.nature.com/nature/journal/v456/n7218/full/456045a.html

149. Laederach, A., Das, R., Vicens, Q, Pearlman, S, Brenowitz, M., Herschlag, D., and Altman, R. (2008) Nature Protocols 3, 1395-1401. Semi-automated and Rapid Quantification of Nucleic Acid Footprinting and Structure Mapping Experiments. (Medline) (PDF File)

148. O'Brien, P., Lassila, J., Fenn, T., Zalatan, J. and Herschlag, D. (2008) Biochemistry 47, 7663-7672. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in E. coli Alkaline Phosphatase. (Medline) (PDF File)

147. Chu, V.B. and Herschlag, D. (2008) Curr. Opin. Struc. Biol. 18, 305-314. Unwinding RNA’s Secrets: Advances in the Biology, Physics, and Modeling of Complex RNAs. (Medline) (PDF File)

146. Forconi, M., Lee, J., Lee, J., Piccirilli, J., and Herschlag, D. (2008) Biochemistry 47, 6883-6894. Functional Identification of Ligands for a Catalytic Metal Ion in Group I Introns. (Medline) (PDF File)

145. Bai, Y., Chu, V.B., Lipfert, J., Pande, V.S., Herschlag, D. and Doniach, S. (2008) J. Am. Chem. Soc. 130, 12334-12341. Critical Assessment of Nucleic Acid Electrostatics via Experimental and Computational Investigation of an Unfolded State Ensemble. PMCID in process (Medline) (PDF File)
See also: Faculty of 1000 Biology, Dec 2008 http://www.f1000biology.com/article/id/1128869

144. Mueller-Planitz, F. and Herschlag, D. (2008) J. Biol. Chem. 283, 17463-17476. Coupling between ATP Binding and DNA Cleavage by DNA Topoisomerase II: A Unifying Kinetic and Structural Mechanism. PMC2427340 (Medline) (PDF File)

143. Sattin, B.D., Zhao, W., Travers, K., Chu, S. and Herschlag, D. (2008) J. Am. Chem. Soc. 130, 6085-6087. Direct Measurement of Tertiary Contact Cooperativity in RNA Folding. PMCID in process (Medline) (PDF File)
See also: “Navigating the RNA Folding Landscape” Nature Chemical Biology 4, 451-452 (2008). http://www.nature.com/nchembio/journal/v4/n8/full/nchembio0808-451.html

142. Hendrickson, D.G., Hogan, D.J., Herschlag, D. Ferrell, J.E. and Brown, P.O. (2008) PloS One 3, e2126. Systematic Identification of mRNAs Recruited to RISC by Specific microRNAs and Corresponding Changes in Transcript Abundance. (Medline) (PDF File)

141. Das, R., Kudaravalli, M., Jonikas, M., Laederach, A., Fong, R., Schwans, J.P., Baker, D., Piccirilli, J.A., Altman, R.B. and Herschlag, D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 4144-4149. Structural Inference of Native and Partially Folded RNA by High-Throughput Contact Mapping. (Medline) (PDF File)
See also: SeparationsNow.com, April 2008 http://www.separationsnow.com/coi/cda/detail.cda?id=18511&type=Feature&chId=2&page=1
Faculty of 1000 Biology, 17 Sept. 2008 http://www.f1000biology.com/article/id/1119588/evaluation

2007

140. Bai, Y., Greenfeld, M., Travers, K., Chu, V.B., Lipfert, J., Doniach, S. and Herschlag, D. (2007) J. Am. Chem. Soc. 129, 14981-14988. Quantitative and Comprehensive Decomposition of the Ion Atmosphere around Nucleic Acids. (Medline) (PDF File)

139. Sigala, P.A., Fafarman, A.T., Bogard, P.E., Boxer, S.G. and Herschlag, D. (2007) J. Am. Chem. Soc. 129, 12104-12105. Do Ligand Binding and Solvent Exclusion Alter the Electrostatic Character within the Oxyanion Hole of an Enzymatic Active Site? (Medline) (PDF File) See also: Faculty of 1000 Biology, 16 Oct. 2007 http://www.f1000biology.com/article/id/1091226/evaluation

138. Forconi, M., Piccirilli, J.A. and Herschlag, D.  (2007) RNA 13, 1656-1667. Modulation of Individual Steps in Group I Intron Catalysis by a Peripheral Metal Ion. (Medline) (PDF File)

137. Chu, V.B., Bai, Y., Lipfert, J., Herschlag, D. and Doniach, S. (2007) Biophysical Journal 9, 3202-3209 Evaluation of Ion Binding to DNA Duplexes Using a Size-Modified Poisson-Boltzmann Theory. (Medline) (PDF File)

136. Zalatan, J.G., Catrina, I., Mitchell, R., Grzyska, P.K., O'Brien, P.J., Herschlag, D. and Hengge, A.C. (2007) J. Am. Chem. Soc. 129, 9789-9798. Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active Site Metal Ions in Catalysis. (Medline) (PDF File)

135. Travers, K., Boyd, N. and Herschlag, D. (2007) RNA 13, 1205-1213. Low Specificity of Metal Ion Binding in the Metal Ion Core of a Folded RNA. (Medline) (PDF File)

134. Mueller-Planitz, F., and Herschlag, D. (2007) Nucleic Acids Research 35, 3764-3773. DNA Topoisomerase II Selects DNA Cleavage Sites Based on Reactivity Rather than Binding Affinity. (Medline) (PDF File)

133. Karbstein, K., Lee, J. and Herschlag, D. (2007) Biochemistry 46, 4861-4875. Probing the Role of a Secondary Structure Element at the 5'- and 3'-Splice Sites in Group I Intron Self-splicing: The L-16 ScaI Ribozyme Reveals a New Role of the G•U Pair in Self-splicing. (Medline) (PDF File)

132. Catrina, I., O'Brien, Purcell, J., Nikolic-Hughes, I., Zalatan, J.G., Hengge, A.C., and Herschlag, D. (2007) J. Am. Chem. Soc. 129, 5760-5765 Probing the Origin of the Compromised Catalysis of E. coli Alkaline Phosphatase in its Promiscuous Sulfatase Reaction. (Medline) (PDF File)

131. Lee, T.H., Lapidus, L.J., Zhao, W., Travers, K.J., Herschlag, D. and Chu, S. (2007) Biophysical Journal 92, 3275-3283. Measuring the Folding Transition Time of Single RNA Molecules. (Medline) (PDF File) See also: Faculty of 1000 Biology, 30 Jan. 2009 http://www.f1000biology.com/article/id/1147123

130. Lipfert, J., Chu, V.B., Bai, Y., Herschlag, D. and Doniach, S. (2007) Journal of Applied Crystallography 40, 235-239 Low Resolution Models for Nucleic Acids from Small-Angle X-ray Scattering with Applications to Electrostatic Modeling. (PDF File)

129. Lipfert, J., Das, R., Chu, V.B., Kudaravalli, M., Boyd, N., Herschlag, D. and Doniach, S. (2007) J. Mol. Biol. 365, 1393-1406. Structural Transitions and Thermodynamics of a Glycine-Dependent Riboswitch from Vibrio cholerae. (Medline) (PDF File)

2006

128. Woodside, M.T., Anthony, P.C., Behnke-Parks, W., Larizadeh, K., Herschlag, D. and Block, S.M. (2006) Science 314, 1001-1004. Direct Measurement of the Full, Sequence-dependent Folding Landscape of a Nucleic Acid. (Medline) (PDF File)

127. Fierke, C.A. and Herschlag, D. (2006) Curr. Opin. Chem. Biol. 10, 453-454. The Wide Reach of Enzymology: From Bioorganic Chemistry to Chemical Biology and Beyond. Editorial Overview.  (PDF File)

126. Russell, R., Das, R., Suh, H., Travers, K., Laederach, A., Engelhardt, M. and Herschlag, D. (2006) J. Mol. Biol. 363, 531-544. The Paradoxical Behavior of a Highly Structured Misfolded Intermediate in RNA Folding. (Medline) (PDF File)

125. Mueller-Planitz, F. and Herschlag, D. (2006) J. Biol. Chem. 281, 23395-23404. Interdomain Communication in DNA Topoisomerase II: DNA Binding and Enzyme Activation. (Medline) (PDF File)

124. Zalatan, J.G., Fenn, T.D., Brunger, A.T., and Herschlag, D. (2006) Biochemistry 45, 9788-9803. Structural and Functional Comparisons of Nucleotide Pyrophosphatase/Phosphodiesterase and Alkaline Phosphatase: Implications for Mechanism and Evolution. (Medline) (PDF File)

123. Woodside MT, Behnke-Parks WM, Larizadeh K, Travers K, Herschlag D, Block SM. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 6190-6195. Nanomechanical Measurements of the Sequence-dependent Folding Landscapes of Single Nucleic Acid Hairpins. (Medline) (PDF File)

122. Kraut, D.A., Sigala, P.A., Pybus, B., Liu, C.W., Ringe, D., Petsko, G.A. and Herschlag, D. (2006) PloS Biology 4 e99. Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole. (Medline) (PDF File)

121. Gerber, A.P., Luschnig, S., Krasnow, M.A., Brown, P.O. and Herschlag, D. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 4487-4492. Genome-wide Identification of mRNAs Associated with the Translational Regulator PUMILIO in Drosophila melanogaster. (Medline) (PDF File)

120. Zalatan, J.G. and Herschlag, D. (2006) J. Am. Chem. Soc. 128, 1293-1303. Alkaline Phosphatase Mono- and Di-esterase Reactions: Comparative Transition State Analysis. (Medline) (PDF File)

2005

119. Hougland, J., Piccirilli, J., Forconi, M., Lee, J. and Herschlag, D. (2005) RNA World 3rd Edition How the Group I Intron Works: A Case Study of RNA Structure and Function. Gesteland, R.F., Cech, T.R. and Atkins, J.F., Editors. Cold Spring Harbor Laboratory Press, New York. pp. 133-205. (PDF File)

118. Johnson, T.H., Tijerina, P., Chadee, A.B., Herschlag, D. and Russell, R. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 10176-10181 Structural Specificity Conferred by a Group I RNA Peripheral Element. (Medline) (PDF File)

117. Das, R., Travers, K., Bai, Y. and Herschlag, D. (2005) J. Am. Chem. Soc. 127, 8272-8273 Determining the Mg²⁺ Stoichiometry for Folding an RNA Metal Ion Core. (Medline) (PDF File)
See also: Faculty of 1000, 21 June 2005 http://www.f1000biology.com/article/id/1011707

116. Nikolic-Hughes, I., O'Brien, P.J. and Herschlag, D. (2005) J. Am. Chem. Soc. 127, 9314–9315 Alkaline Phosphatase Catalysis Is Ultrasensitive to Charge Sequestered Between the Active Site Zinc Ions. (Medline) (PDF File)

115. Forconi, M. and Herschlag, D. (2005) J. Am. Chem. Soc. 127, 6160-6161 Promiscuous Catalysis by the Tetrahymena Group I Ribozyme. (Medline) (PDF File)

114. Hougland, J.L., Kravchuk, A.V., Herschlag, D. and Piccirilli, J.A. (2005) PLoS Biology 3 e277 Functional Identification of a Catalytic Metal Ion Binding Site Within RNA. (Medline) (PDF File)

113. Arava, Y., Boas, F.E., Brown, P.O. and Herschlag, D. (2005) Nucleic Acids Research 33, 2421-2432 Dissecting Eukaryotic Translation and Its Control By Ribosome Density Mapping. (Medline) (PDF File)

112. Bai, Y., Das, R., Millet, I.S., Herschlag, D. and Doniach, S. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 1035-1040. Probing Counterion Modulated Repulsion and Attraction Between Nucleic Acid Duplexes in Solution. (Medline) (PDF File)

111. Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. and Altman, R.B. (2005) RNA 11, 344-354 SAFA: Semi-Automated Footprinting Analysis Software for High-throughput Quantification of Nucleic Acid Footprinting Experiments. (Medline) (PDF File)

2004

110. Andresen, K., Das, R., Park, H.Y., Smith, H., Kwok, L.W., Lamb, J.S., Kirkland, E.J., Herschlag, D., Finkelstein, K.D. and Pollack, L. (2004) Physical Review Letters 93, 248103-1 - 248103-4. Spatial Distribution of Competing Ions Around DNA in Solution. (Medline) (PDF File)

109. Karbstein, K., Tang, K-H. and Herschlag, D. (2004) RNA 10, 1730-1739. A Base Triple in the Tetrahymena Group I Core Affects the Reaction Equilibrium via a Threshold Effect. (Medline) (PDF File)

108. Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D. and Chance, M. (2004) J. Mol. Biol. 343, 1195-1206. Principles of RNA Compaction: Insights From the Equilibrium Folding Pathway of the P4-P6 RNA Domain in Monovalent Cations. (Medline) (PDF File)

107. Nikolic-Hughes, I., Rees, D. C., Herschlag, D. (2004) J. Am. Chem. Soc. 126, 11814-11819. Do Electrostatic Interactions with Positively Charged Active Site Groups Tighten the Transition State for Enzymatic Phosphoryl Transfer? (Medline) (PDF File)

106. Gerber, A.P., Herschlag, D. and Brown, P.O. (2004) PLoS Biology 2, 342-354. Extensive Association of Functionally and Cytotopically Related mRNAs with Puf-family RNA-binding Proteins in Yeast. (Medline) (PDF File)

2003

105. Shepard, K.A., Gerber, A.P., Jambhekar, A., Takizawa, P.A., Herschlag, D., Brown, P.O., DeRisi, J.L. and Vale, R.D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11429-11434. Widespread Cytoplasmic mRNA Transport in S. cerevisiae: Identification of 20 New Bud-localized Transcripts Using DNA Microarray Analysis. (Medline) (PDF File)

104. Das, R., Kwok, L.W., Millet, I.S., Bai, Y., Mills, T.T., Jacob, J., Maskel, G.S., Seifert, S., Simon, M.G.J., Thiyagarajan, P., Doniach, S., Pollack, L. and Herschlag, D. (2003) J. Mol. Biol. 332, 311-319. The Fastest Global Events in RNA Folding: Electrostatic Relaxation and Tertiary Collapse of the Tetrahymena Ribozyme. (Medline) (PDF File)

103. Peck, M.L. and Herschlag, D. (2003) RNA 9, 1180-1187. Adenosine 5’-O-(3-thio) Triphosphate (ATPS) Is a Substrate for the Nucleotide Hydrolysis and RNA Unwinding Activities of Eukaryotic Translation Initiation Factor eIF4A. (Medline) (PDF File)

102. Das, R., Mills, T.T., Kwok, L.W., Maskel, G.S., Millett, I.S., Doniach, S., Finkelstein, K.D., Herschlag, D. and Pollack, L. (2003) Physical Review Letters 90, 188103-1 - 188103-4. The Counterion Distribution Around DNA Probed by Solution X-Ray Scattering. (Medline) (PDF File)

101. Kraut, D.A., Carroll, K.S. and Herschlag, D. (2003) Annu. Rev. Biochem. 72, 517-571. Challenges in Enzyme Mechanism and Energetics. (Medline) (PDF File)

100. Bartley, L.E., Zhuang, X., Das, R., Chu, S. and Herschlag, D. (2003) J. Mol. Biol. 328, 1011-1026. Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a Large Structured RNA. (Medline) (PDF File)

99. Arava, Y., Wang, Y., Storey, J.D., Liu, C.L., Brown, P. and Herschlag, D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 3889-3894. Genome-wide Analysis of mRNA Translation Profiles in Saccharomyces cerevisiae. (Medline) (PDF File)

98. Karbstein, K. and Herschlag, D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2300-2305. Extraordinarily Slow Binding of Guanosine to the Tetrahymena Group 1 Ribozyme. (Medline) (PDF File)

2002

97. Cheng, H., Nikolic-Hughes, I., Wang, J.H., Deng, H., O’Brien, P.J., Wu, L., Zhang, Z.Y., Herschlag, D. and Callender, R. (2002) J. Am. Chem. Soc. 124, 11295-11306. Environmental Effects on Phosphoryl Group Bonding Probed by Vibrational Spectroscopy: Implications for Understanding Phosphoryl Transfer and Enzymatic Catalysis. (Medline) (PDF File)

96. Karbstein, K., Carroll, K.S. and Herschlag, D. (2002) Biochemistry 41, 11171-83. Probing the Tetrahymena Ribozyme Reaction in Both Directions. (Medline) (PDF File)

95. Wang, Y., Liu, C.L., Storey, J.D., Tibshirani, R.J., Herschlag, D. and Brown, P.O. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5860-5865. Precision and Functional Specificity in mRNA Decay. (Medline) (PDF File)

94. Shan, S. and Herschlag, D. (2002) RNA 8, 861-872. Dissection of a Metal Ion Mediated Conformational Change in Tetrahymena Ribzoyme Catalysis. (Medline) (PDF File)

93. Sorin, E.J., Engelhardt, M.A., Herschlag, D. and Pande, V.S. (2002) J. Mol. Biol. 317, 493-506. RNA Simulations: Probing Hairpin Unfolding and the Dynamics of a GNRA Tetraloop. (Medline) (PDF File)

92. Russell, R., Millett, I.S., Tate, M.W., Kwok, L.W., Nakatani, B., Gruner, S.M., Mochrie, S.G.J., Pande, V., Doniach, S., Herschlag, D. and Pollack, L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 4266-4271. Rapid Compaction During RNA Folding. (Medline) (PDF File)

91. O’Brien, P. and Herschlag, D. (2002) Biochemistry 41, 3207-3225. Alkaline Phosphatase Revisited: The Hydrolysis of Alkyl Phosphates. (Medline) (PDF File)

90. Russell, R., Zhaung, X., Babcock, H.P., Millett, I.S., Doniach, S., Chu, S. and Herschlag, D. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 155-160. Exploring the Folding Landscape of a Structured RNA.

(Medline) (PDF File)

2001

89. Peluso, P., Shan, S., Nock, S., Herschlag, D. and Walter, P. (2001) Biochemistry 40, 15224-15233. Role of SRP RNA in the GTPase Cycles of Fth and FtsY. (Medline) (PDF File)

88. Russell, R. and Herschlag, D. (2001) J. Mol. Biol. 308, 839-851. Probing the Folding Landscape of the Tetrahymena Ribozyme: Commitment to Form the Native Conformation Is Late in the Folding Pathway. (Medline) (PDF File)

87. O’Brien, P. and Herschlag, D. (2001) Biochemistry 40, 5691-5699. Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of E. coli Alkaline Phosphatase. (Medline) (PDF File)

86. Shan, S., Kravchuk, A.V., Piccirilli, J.A. and Herschlag, D. (2001) Biochemistry 40, 5161-5171. Defining the Catalytic Metal Ion Interactions in the Tetrahymena Ribozyme Reaction. (Medline) (PDF File)

85. O’Rear , J., Wang, S., Feig, A.L., Uhlenbeck, O.C. and Herschlag, D. (2001) RNA 7, 537-545. Comparison of the Hammerhead Cleavage Reactions Stimulated by Monovalent and Divalent Cations. (Medline) (PDF File)

84. Admiraal, S.J., Meyer, P., Schneider, B., Deville-Conne, D., Janin, J. and Herschlag, D. (2001) Biochemistry 40, 403-413. Chemical Rescue of Phosphoryl Transfer in a Cavity Mutant of Nucleoside Diphosphate Kinase: Implications for Site-Directed Mutagenesis. (Medline)(PDF File)

2000

83. Zhuang, X., Bartley, L.E., Babcock, H.P., Russell, R., Ha, T., Herschlag, D., Chu, S. (2000) Science 288, 2048-2051. A Single Molecule Study of RNA Catalysis and Folding. (Medline)(PDF File)

82. Shan, S., Herschlag, D. (2000) RNA 6, 795-813. An Unconventional Origin of Metal Ion Rescue and Inhibition in the Tetrahymena Group I Ribozyme Reaction. (Medline)(PDF File)

81. Russell, R., Millett, I.S., Doniach, S., Herschlag, D. (2000) Nature Struc. Biol. 5, 367-370. Small Angle X-ray Scattering Reveals a Compact Intermediate in RNA Folding. (Medline)(PDF File)

80. Peluso, P., Herschlag, D., Freymann, D.M., Johnson, A.E., Walter, P. (2000) Science 288, 1640-1643. Role of 4.5S RNA in Assembly of the Bacterial Signal Recognition Particle with Its Receptor. (Medline)(PDF File)

79. Narlikar, G.J., Bartley, L.E., Herschlag, D. (2000) Biochemistry 39, 6183-6189. Use of Duplex Rigidity for Stability and Specificity in RNA Tertiary Structure. (Medline)(PDF File)

78. Admiraal, S.J., Herschlag, D. (2000) J. Am. Chem. Soc. 122, 2145-2148. The Substrate-assisted General Base Catalysis Model for Phosphate Monoester Hydrolysis: Evaluation Using Reactivity Comparisons. (PDF File)

77. Engelhardt, M.A., Doherty, E.A. Knitt, D.S., Doudna, J.A., Herschlag, D. (2000) Biochemistry 39, 2639-2651. The P5abc Peripheral Element Facilitates Preorganization of the Tetrahymena Group I Ribozyme for Catalysis. (Medline)(PDF File)

76. Yoshida, A., Shan, S., Herschlag, D., Piccirilli, J. (2000) Chemistry and Biology 7, 85-96. The Role of the Cleavage Site 2'-Hydroxyl in the Tetrahymena Group I Ribozyme Reaction. (Medline)(PDF File)

1999

75. Lorsch, J. and Herschlag, D. (1999) EMBO J. 18, 6705-6717. Kinetic Dissection of Fundamental Processes of Eukaryotic Translation Initiation In Vitro. (Medline)(PDF File)

74. O’Brien, P. and Herschlag, D. (1999) J. Am. Chem. Soc. 121, 11022-11023. Does the Active Site Arginine Change the Nature of the Transition State for Alkaline Phosphatase-catalyzed Phosphoryl Transfer? (PDF File)

73. Gerton, J., Herschlag, D. and Brown, P. (1999) J. Biol. Chem. 274, 33480-33487. Stereospecificity of Reactions Catalyzed by HIV-1 Integrase. (Medline)(PDF File)

72. Narlikar, G.J., Bartley, L.E., Khosla, M. and Herschlag, D. (1999) Biochemistry 38, 14192-14204, Characterization of a Local Folding Event of the Tetrahymena Group I Ribozyme: Effects of Oligonucleotide Substrate Length, pH, and Temperature on the Two Substrate Binding Steps. (Medline)(PDF File)

71. Shan, S., Yoshida, A., Piccirilli, J. and Herschlag, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 12299-12304. Three Metal Ions at the Active Site of the Tetrahymena Group I Ribozyme. (Medline)(PDF File)

70. Wang, S., Karbstein, K., Peracchi, A., Beigelman, L. and Herschlag, D. (1999) Biochemistry 43, 14363-14278. Identification of the Hammerhead Ribozyme Metal Ion Binding Site Responsible for Rescue of the Deleterious Effect of a Cleavage Site Phosphorothioate. (Medline)(PDF File)

69. Zhang, Y.L., Hollfelder, F., Gordon, S.J., Chen, L., Keng, Y.F., Wu, L., Herschlag, D. and Zhang, Z.Y. (1999) Biochemistry 38, 12111-12123. Impaired Transition State Complementarity in the Hydrolysis of O-Arylphosphorothioates by Protein-Tyrosine Phosphatases. (Medline)(PDF File)

68. Russell, R. and Herschlag, D. (1999) J. Mol. Biol. 291, 1155-1167. New Pathways in Folding of the Tetrahymena Group I RNA Enzyme. (Medline)(PDF File)

67. Peck, M. and Herschlag, D. (1999) RNA 5, 1210-1221. Effects of Oligonucleotide Length and Atomic Composition on Stimulation of the ATPase Activity of Translation Initiation Factor eIF4A. (Medline)(PDF File)

66. Shan, S., Narlikar, G.J. and Herschlag, D. (1999) Biochemistry 38, 10976-10988. Protonated 2’-Aminoguanosine as a Probe for the Electrostatic Environment of the Active Site of the Tetrahymena Group I Ribozyme. (Medline)(PDF File)

65. Shan, S. and Herschlag, D. (1999) Biochemistry 38, 10958-10975. Probing the Role of Metal Ions in RNA Catalysis: Kinetic and Thermodynamic Characterization of Metal Ion Interaction with the 2’-Moiety of the Guanosine Nucleophile in the Tetrahymena Group I Ribozyme. (Medline)(PDF File)

64. Admiraal, S.J. and Herschlag, D. (1999) J. Am. Chem. Soc. 121, 5837-5845. Catalysis of Phosphoryl Transfer from ATP by Amine Nucleophiles. (PDF File)

63. Admiraal, S.J., Schneider, B., Meyer, P., Janin, J., Véron, M., Deville-Bonne, D. and Herschlag, D. (1999) Biochemistry 38, 4701-4711. Nucleophilic Activation by Positioning in Phosphoryl Transfer Catalyzed by Nucleoside Diphosphate Kinase. (Medline)(PDF File)

62. O’Brien, P. and Herschlag, D. (1999) Chemistry and Biology 6, R91-R105. Catalytic Promiscuity and the Evolution of New Enzymatic Activities. (Medline) (PDF File)

61. Doherty, E.A., Herschlag, D. and Doudna, J.A. (1999) Biochemistry 38, 2982 -2990. Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme. (Medline) (PDF File)

60. Korber, P., Zander, T., Herschlag, D. and Bardwell, J.C.A. (1999) J. Biol. Chem. 274, 249-256. A New Heat Shock Protein that Binds Nucleic Acids. (Medline)(PDF File)

59. Russell, R. and Herschlag, D. (1999) RNA 5, 158-166. Specificity from Steric Restrictions in the Guanosine Binding Pocket of a Group I Ribozyme. (Medline)(PDF File)

58. Shan, S. and Herschlag, D. (1999) Hydrogen Bonding in Enzymatic Catalysis: Analysis of Energetic Contributions. In Methods in Enzymology, Vol. 308, Schramm V.L. and Purich, D.L., eds. Academic Press, San Diego. pp. 246-276. (Medline)(PDF File)

1998

57. O’Brien, P. and Herschlag, D. (1998) J. Am. Chem. Soc. 120, 12369-12370. Sulfatase Activity of E. coli Alkaline Phosphatase Demonstrates a Functional Link to Arylsulfatases, an Evolutionarily Related Enzyme Family. (PDF File)

56. Herschlag, D. (1998) Nature 395, 548-549. Ribozyme Crevices and Catalysis (News and Views).

(Medline)(PDF File)

55. Peracchi, A., Beigelman, L., Karpeisky, A., Maloney, L. and Herschlag, D. (1998) Biochemistry 37, 14765-14775. A Core Folding Model for Catalysis by the Hammerhead Ribozyme Accounts for its Extraordinary Sensitivity to Abasic Mutations. (Medline)(PDF File)

54. Peracchi, A., Matulic-Adamic, J., Wang, S.L., Beigelman, L., Herschlag, D. (1998) RNA 4, 1332-1346. Structure-Function Relationships in the Hammerhead Ribozyme Probed by Base Rescue. (Medline)(PDF File)

53. Narlikar, G.J., Herschlag, D. (1998) Biochemistry 37, 9902-9911. Direct Demonstration of the Catalytic Role of Binding Interactions in Enzymatic Reactions. (Medline)(PDF File)

52. Lorsch, J.R., Herschlag, D. (1998) Biochemistry 37, 2194-2206. The DEAD Box Protein eIF4A. 2. A Cycle of Nucleotide and RNA-dependent Conformational Changes. (Medline)(PDF File)

51. Lorsch, J.R. and Herschlag, D. (1998) Biochemistry 37, 2180-2193. The DEAD Box Protein eIF4A. 1. A Kinetic and Thermodynamic Framework Reveals Coupled Binding of RNA and Nucleotide. (Medline)(PDF File)

1997

50. Peracchi, A., Beigelman, L., Scott, E.C., Uhlenbeck, O.C., Herschlag, D. (1997) J. Biol. Chem. 272, 26822-26826. Involvement of a Specific Metal Ion in the Transition of the Hammerhead Ribozyme to Its Catalytic Conformation. (Medline)(PDF File)

49. Hertel, K.J., Peracchi, A., Uhlenbeck, O.C., Herschlag, D. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 8497-8502. Use of Intrinsic Binding Energy for Catalysis by an RNA Enzyme. (Medline)(PDF File)

48. McConnell, T.S., Herschlag, D., Cech, T.R. (1997) Biochemistry 36, 8293-8303. The Effects of Divalent Metal Ions on Individual Steps of the Tetrahymena Ribozyme Reaction. (Medline)(PDF File)

47. Narlikar, G.J., Herschlag, D. (1997) Annu. Rev. Biochem. 66,19-59. Mechanistic Aspects of Enzymatic Catalysis: Lessons from Comparison of RNA and Protein Enzymes. (Medline)(PDF File)

46. Narlikar, G.J., Khosla, M., Usman, N., Herschlag, D. (1997) Biochemistry 36, 2465-2477. Quantitating Tertiary Binding Energies of 2' Hydroxyl Groups on the P1 Duplex of the Tetrahymena Ribozyme: Intrinsic Binding Energy in an RNA Enzyme. (Medline)(PDF File)

45. Shan, S. and Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 14474-14479. The Change in Hydrogen Bond Strength Accompanying Charge Rearrangement: Implications for Enzymatic Catalysis.(Medline) (PDF File)

44. Peracchi, A., Beigelman, L., Usman, N. and Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522-11527. Rescue of Abasic Hammerhead Ribozymes by Exogenous Addition of Specific Bases. (Medline) (PDF File)

43. Narlikar, G.J. and Herschlag, D. (1996) Nature Struc. Biol. 3, 701-710. Isolation of a Local Tertiary Folding Transition in the Context of a Globally Folded RNA. (Medline) (PDF File)

1996

42. Maegley, K.A., Admiraal, S.J. and Herschlag, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8160-8166. Ras-catalyzed Hydrolysis of GTP: Insights from Model Studies. (Medline)(PDF File)

41. Shan, S. and Herschlag, D. (1996) J. Am. Chem. Soc. 118, 5515-5518. Energetic Effects of Multiple Hydrogen Bonds. Implications for Enzymatic Catalysis. (PDF File)

40. Hertel, K.J., Herschlag, D. and Uhlenbeck, O.C. (1996) EMBO J. 15, 3751-3757. Specificity of Hammerhead Ribozyme Cleavage. (Medline)(PDF File)

39. Cech, T.R. and Herschlag, D. (1996) Group I Ribozymes: Substrate Recognition, Catalytic Strategies and Comparative Mechanistic Analysis. In Nucleic Acids and Molecular Biology: Special Issue on RNA Catalysi, Vol. 10, Eckstein, F. and Lilley, D.M.J., Editors. Springer-Verlag, Berlin, pp 1-17. (PDF File)

38. Mei, R. and Herschlag, D. (1996) Biochemistry 35, 5796-5809. Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron. Insights into Catalysis and the Second Step of Self-Splicing. (Medline)(PDF File)

37. Shan, S., Loh, S. and Herschlag, D. (1996) Science 272, 97-101. The Energetics of Hydrogen Bonds in Model Systems. Implications for Enzymatic Catalysis. (Medline)(PDF File)

36. Knitt, D.S. and Herschlag, D. (1996) Biochemistry 35, 1560-1570. pH Dependencies of the Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent pK^a. (Medline)(PDF File)

1995

35. Admiraal, S.J., Herschlag, D. (1995) Chemistry and Biology 2, 729-739. Mapping the Transition State for ATP Hydrolysis. Implications for Enzymatic Catalysis. (Medline)(PDF File)

34. Hollfelder, F., Herschlag, D. (1995) Biochemistry 34, 12255-12264. The Nature of the Transition State for Enzyme-catalyzed Phosphoryl Transfer. Hydrolysis of O-Arylphosphorothioates by Alkaline Phosphatase. (Medline)(PDF File)

33. Herschlag, D. (1995) J. Biol. Chem. 270, 20871-20874. RNA Chaperones and the RNA Folding Problem. (Medline)(PDF File)

32. Narlikar, G.J., Gopalakrishnan, V., McConnell, T.S., Usman, N., Herschlag, D. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3668-3672. Use of Binding Energy by an RNA Enzyme for Catalysis by Positioning and Substrate Destabilization. (Medline)(PDF File)

1994

31. Herschlag, D. (1994) J. Am. Chem. Soc. 116, 11631-11635. Ribonuclease Revisited: Catalysis Via the Classical General Acid-Base Mechanism or a Triester-like Mechanism? (PDF File)

30. Knitt, D.S., Narlikar, G.J., Herschlag, D. (1994) Biochemistry 33, 13864-13879. Dissection of the Role of the Conserved G•U Pair in Group I RNA Self-splicing. (Medline)(PDF File)

29. Coetzee, T., Herschlag, D., Belfort, M. (1994) Genes & Development 8, 1575-1588. Escherichia Coli Proteins, Including Ribosomal Protein S12 Facilitate In Vitro Splicing of Phage T4 Introns by Acting as RNA Chaperones. (Medline)(PDF File)

28. Herschlag, D., Khosla, M., Tsuchihashi, Z., Karpel, R.L. (1994) EMBO J. 13, 2913-2924. An RNA Chaperone Activity of Nonspecific RNA Binding Proteins in Hammerhead Ribozyme Catalysis.

(Medline)(PDF File)

27. Herschlag, D., Khosla, M. (1994) Biochemistry 33, 5291-5297. Comparison of pH Dependencies of the Tetrahymena Ribozyme Reactions with RNA 2’-Substituted and Phosphorothioate Substrates Reveals a Rate-limiting Conformational Step. (Medline)(PDF File)

26. Hertel, K. J., Herschlag, D. and Uhlenbeck, O.C. (1994) Biochemistry 33, 3374-3385. A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction. (Medline)(PDF File)

1993

25. Tsuchihashi, Z., Khosla, M. and Herschlag, D. (1993) Science 262, 99-102. Protein Enhancement of Hammerhead Ribozyme Catalysis. (Medline) (PDF File)

24. McConnell, T.S., Cech, T.R. and Herschlag, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8362-8366. Guanosine Binding to the Tetrahymena Ribozyme: Thermodynamic Coupling with Oligonucleotide Binding. (Medline) (PDF File)

23. Herschlag, D. and Johnson, F.B. (1993) Genes & Development 7, 173-179. Synergism in Transcriptional Activation: A Kinetic View. (Medline) (PDF File)

The above publications are as an Independent Investigator (7/92-Present)

22. Labow, B., Herschlag, D. and Jencks, W.P. (1993) Biochemistry 32, 8737-8741. Catalysis of the Hydrolysis of Phosphorylated Pyridines by Alkaline Phosphatase Has Little or No Dependence on the pK^a of the Leaving Group. (Medline) (PDF File)

21. Herschlag, D., Eckstein, F. and Cech, T.R. (1993) Biochemistry 32, 8312-8321. The Importance of Being Ribose at the Cleavage Site in the Tetrahymena Ribozyme Reaction. (Medline) (PDF File)

20. Herschlag, D., Eckstein, F. and Cech, T.R. (1993) Biochemistry 32, 8299-8311. Contributions of 2’ Hydroxyl Groups of the RNA Substrate to Binding and Catalysis by the Tetrahymena Ribozyme. An Energetic Picture of an Active Site Composed of RNA. (Medline) (PDF File)

19. Legault, P., Herschlag, D., Celander, D.W. and Cech, T.R. (1992) Nucleic Acids Res. 20, 6613-6619. Mutations at the Guanosine-binding Site of the Tetrahymena Ribozyme Also Affect Site-specific Hydrolysis. (Medline) (PDF File)

18. Cech. T.R., Herschlag, D., Piccirilli, J.A. and Pyle, A.M. (1992) J. Biol. Chem. 267, 17479-17482. RNA Catalysis by a Group I Ribozyme. Developing a Model for Transition State Stabilization. (Medline) (PDF File)

17. Herschlag, D. (1992) Biochemistry 31, 1386-1399. Evidence for Processivity and Two-Step Binding of the RNA Substrate From Studies of J1/2 Mutants of the Tetrahymena Ribozyme. (Medline) (PDF File)

16. Young, B., Herschlag, D. and Cech, T.R. (1991) Cell 67, 1007-1019. Mutations in a Nonconserved Sequence of the Tetrahymena Ribozyme Increase Activity and Specificity. (Medline) (PDF File)

15. Herschlag, D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6921-6925. Implications of Ribozyme Kinetics for Targeting the Cleavage of Specific RNA Molecules In Vivo: More Isn't Always Better. (Medline) (PDF File)

14. Herschlag, D., Piccirilli, J.A. and Cech, T.R. (1991) Biochemistry 30, 4844-4854. Ribozyme-catalyzed and Non-enzymatic Reactions of Phosphate Diesters: Rate Effects upon Substitution of Sulfur for a Non-bridging Phosphoryl Oxygen Atom. (Medline) (PDF File)

13. Herschlag, D. and Cech, T.R. (1990) Biochemistry 29, 10172-10180. Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme. 2. Kinetic Description of the Reaction of an RNA Substrate that Forms a Mismatch at the Active Site. (Medline) (PDF File)

12. Herschlag, D. and Cech, T.R. (1990) Biochemistry 29, 10159-10171. Catalysis of RNA Cleavage by the Tetrahymena thermophila Ribozyme. 1. Kinetic Description of the Reaction of an RNA Substrate Complementary to the Active Site. (Medline) (PDF File)

11. Herschlag, D. and Cech, T.R. (1990) Nature 344, 405-409. DNA Cleavage Catalysed by the Ribozyme from Tetrahymena. (Medline) (PDF File)

10. Herschlag, D. and Jencks, W.P. (1990) Biochemistry 29, 5172-5179. Catalysis of the Hydrolysis of Phosphorylated Pyridines by Mg(OH)⁺: A Possible Model for Enzymatic Phosphoryl Transfer. (Medline) (PDF File)

9. Herschlag, D. and Jencks, W.P. (1990) J. Am. Chem. Soc.112, 1951-1956. Nucleophiles of High Reactivity in Phosphoryl Transfer Reactions: -Effect Compounds and Fluoride Ion. (PDF File)

8. Herschlag, D. and Jencks, W.P. (1990) J. Am. Chem. Soc. 112, 1942-1950. The Effect of Mg²⁺, Hydrogen Bonding and Steric Factors on Rate and Equilibrium Constants for Phosphoryl Transfer between Carboxylate Ions and Pyridines. (PDF File)

7. Herschlag, D. and Jencks, W.P. (1989) J. Am. Chem. Soc. 111, 7587-7596. Phosphoryl Transfer to Oxyanions: The Nature of the Transition State and Electrostatic Repulsion. (PDF File)

6. Herschlag, D. and Jencks, W.P. (1989) J. Am. Chem. Soc.111, 7579-7586. Evidence That Metaphosphate is Not an Intermediate in Solvolysis Reactions in Aqueous Solution. (PDF File)

5. Herschlag, D. (1988) Bioorganic Chemistry 16, 62-96. The Role of Induced Fit and Conformational Changes of Enzymes in Specificity and Catalysis. (PDF File)

4. Herschlag, D. and Jencks, W.P. (1987) J. Am. Chem. Soc. 109, 4665-4674. The Effect of Divalent Metal Ions on the Rate and Transition State Structure of Phosphoryl Transfer Reactions. (PDF File)

3. Herschlag, D. and Jencks, W.P. (1986) J. Am. Chem. Soc.108, 7938-7946. Pyrophosphate Formation from Acetyl Phosphate and Orthophosphate Anions in Concentrated Aqueous Salt Solutions Does Not Provide Evidence for a Metaphosphate Intermediate. (PDF File)

2. Jencks, W.P., Haber, M.T., Herschlag, D. and Nazaretian, K.L. (1986) J. Am. Chem. Soc. 108, 479-483. Decreasing Reactivity with Increasing Nucleophile Basicity. The Effect of Solvation on βnuc for Phosphoryl Transfer to Amines. (Medline) (PDF File)

1. Herschlag, D., Stevens, E.S. and Gander, J.E. (1983) Int. J. Peptide Prot. Res. 22, 16-20. Galactofuranosyl-containing Glycopeptide of Penicillium charlesii: Vacuum Ultraviolet Circular Dichroism. (Medline)(PDF File)