Nina M. Goodey

Allosteric regulation and catalysis emerge via a common route

Allosteric regulation of protein function is a mechanism by which an event in one place of a protein structure causes an effect at another site, much like the behavior of a telecommunications network in which a collection of transmitters, receivers and transceivers communicate with each other across long distances. For example, ligand binding or an amino acid mutation at an allosteric site can alter enzymatic activity or binding affinity in a distal region such as the active site or a second binding site. The mechanism of this site-to-site communication is of great interest, especially since allosteric effects must be considered in drug design and protein engineering. In this review, conformational mobility as the common route between allosteric regulation and catalysis is discussed. We summarize recent experimental data and the resulting insights into allostery within proteins, and we discuss the nature of future studies and the new applications that may result from increased understanding of this regulatory mechanism.

Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase.

One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H–C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor–acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor–acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface pre-arrangement relative to the WT enzyme. Apparently, the coupling of the enzymes dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.

Catalytic contributions from remote regions of enzyme structure.

Catalytic Contributions from Remote Regions of Enzyme Structure Jeeyeon Lee* and Nina M. Goodey* Department of Chemistry, 413 Wartik Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA College of Pharmacy, Ajou University, San 5, Woncheon-dong, Yeongtong-Gu, Suwon, Korea 443-749 Department of Chemistry and Biochemistry, Montclair State University, 1 Normal Avenue, Montclair, New Jersey 07043, USA

Temporally Overlapped but Uncoupled Motions in Dihydrofolate Reductase Catalysis

Temporal correlations between protein motions and enzymatic reactions are often interpreted as evidence for catalytically important motions. Using dihydrofolate reductase as a model system, we show that there are many protein motions that temporally overlapped with the chemical reaction, and yet they do not exhibit the same kinetic behaviors (KIE and pH dependence) as the catalyzed chemical reaction. Thus, despite the temporal correlation, these motions are not directly coupled to the chemical transformation, and they might represent a different part of the catalytic cycle or simply be the product of the intrinsic flexibility of the protein.

Prediction of residues involved in inhibitor specificity in the dihydrofolate reductase family.

Dihydrofolate reductase (DHFR) is of significant recent interest as a target for drugs against parasitic and opportunistic infections. Understanding factors which influence DHFR homolog inhibitor specificity is critical for the design of compounds that selectively target DHFRs from pathogenic organisms over the human homolog. This paper presents a novel approach for predicting residues involved in ligand discrimination in a protein family using DHFR as a model system. In this approach, the relationship between inhibitor specificity and amino acid composition for sets of protein homolog pairs is examined. Similar inhibitor specificity profiles correlate with increased sequence homology at specific alignment positions. Residue positions that exhibit the strongest correlations are predicted as specificity determinants. Correlation analysis requires a quantitative measure of similarity in inhibitor specificity (S(lig)) for a pair of homologs. To this end, a method of calculating S(lig) values using K(I) values for the two homologs against a set of inhibitors as input was developed. Correlation analysis of S(lig) values to amino acid sequence similarity scores - obtained via multiple sequence alignments - was performed for individual residue alignment positions and sets of residues on 13 DHFRs. Eighteen alignment positions were identified with a strong correlation of S(lig) to sequence similarity. Of these, three lie in the active site; four are located proximal to the active site, four are clustered together in the adenosine binding domain and five on the βFβG loop. The validity of the method is supported by agreement between experimental findings and current predictions involving active site residues.

Views from academia and industry on skills needed for the modern research environment

Reports from employers of higher education graduates indicate the existence of a considerable gap between the skills required by employers and those possessed by recent graduates. As a first step toward closing this gap, this study aims to determine its origin. Interviews with nine research‐active biochemistry professionals were used to identify the most important skills for biochemistry students to succeed in research positions postgraduation. The results of these interviews were used to develop a survey, which was then administered to a larger group of biochemistry faculty and industry professionals. The output of the survey was a list of 52 skills valued by biochemistry professionals and rated by perceived importance. Importantly, the survey results also afford a comparative look at the prioritization of skills by two key populations: the academic faculty training students and the industry professionals hiring them. While there are many areas of agreement between these two populations, the survey also reveals areas were priorities diverge. The discrepancies found here suggest that the skills gap manifest at the point of employment may stem directly from differences in prioritization between the academic and industrial environments. This article aims to provide insight into the needs and requirements of the modern biochemical research environment, and invites debate concerning the preparation students receive in academia. Moreover, the results presented herein point to a need for further exploration of the possible misalignment of these two critical environments for young scientists.

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C(MDCC) DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067μM(-1)s(-1)), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C(MDCC) DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C(MDCC) DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.

Understanding Enzyme Mechanism through Protein Chimeragenesis

The preparation of chimeras, proteins that contain segments from two or more different parent proteins, is a valuable tool in protein engineering yielding structures with novel properties. In addition to the obvious practical value of hybrid proteins as catalysts and biopharmaceuticals, their careful analysis can be used to understand the role of specific domains in enzymatic catalysis and protein evolution in a unique way that complements other structure-function studies. The study of hybrid enzymes can reveal, for example, the role specific subunits and/or domains play in dictating substrate specificity, catalytic activity, processivity, and stability. Popular chimeragenesis methods, including noncombinatorial and combinatorial methods, that can be used to generate hybrid proteins, are discussed here and four case studies are presented that beautifully demonstrate how hybrids can be studied to gain detailed understanding about substrate selectivity, enzymatic activity, S.J. Benkovic Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, USA, e-mail: sjb1@psu.edu

Mobile interaction and query optimizationin a protein-ligand data analysis system

With current trends in integrating phylogenetic analysis into pharma-research, computing systems that integrate the two areas can help the drug discovery field. DrugTree is a tool that overlays ligand data on a protein-motivated phylogenetic tree. While initial tests of DrugTree are successful, it has been noticed that there are a number of lags concerning querying the tree. Due to the interleaving nature of the data, query optimization can become problematic since the data is being obtained from multiple sources, integrated and then presented to the user with the phylogenetic imposed upon the phylogenetic analysis layer. This poster presents our initial methodologies for addressing the query optimization issues. Our approach applies standards as well as uses novel mechanisms to help improve performance time.