Enhanced Conformational Space Sampling Improves the Prediction of Chemical Shifts in Proteins

Markwick, Phineus R. L.; Cervantes, Carla F.; Abel, Barrett L.; Komives, Elizabeth A.; Blackledge, Martin; McCammon, J. Andrew

doi:10.1021/ja9093692

Cited by 81 publications

(80 citation statements)

References 16 publications

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“…S2) at the active site of C o . Similar to our results, aMD has been shown to successfully increase the rate of conformational sampling, thereby characterizing millisecond-timescale protein/peptide dynamical motions and achieving notable agreement with experimental data (25)(26)(27)(28)(29). Our simulations further confirmed recent experimental observations (13,14) that ECD in CypA takes place over a broad range of timescales even in the substrate-free state (Fig.…”

Section: Accelerating Cypa Dynamics and Its Effects On The Chemicalsupporting

confidence: 90%

Resolving the complex role of enzyme conformational dynamics in catalytic function

Doshi

McGowan

Ladani

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Despite growing evidence suggesting the importance of enzyme conformational dynamics (ECD) in catalysis, a consensus on how precisely ECD influences the chemical step and reaction rates is yet to be reached. Here, we characterize ECD in Cyclophilin A, a well-studied peptidyl-prolyl cis-trans isomerase, using normal and accelerated, atomistic molecular dynamics simulations. Kinetics and free energy landscape of the isomerization reaction in solution and enzyme are explored in unconstrained simulations by allowing significantly lower torsional barriers, but in no way compromising the atomistic description of the system or the explicit solvent. We reveal that the reaction dynamics is intricately coupled to enzymatic motions that span multiple timescales and the enzyme modes are selected based on the energy barrier of the chemical step. We show that Kramers' rate theory can be used to present a clear rationale of how ECD affects the reaction dynamics and catalytic rates. The effects of ECD can be incorporated into the effective diffusion coefficient, which we estimate to be about ten times slower in enzyme than in solution. ECD thereby alters the preexponential factor, effectively impeding the rate enhancement. From our analyses, the trend observed for lower torsional barriers can be extrapolated to actual isomerization barriers, allowing successful prediction of the speedup in rates in the presence of CypA, which is in notable agreement with experimental estimates. Our results further reaffirm transition state stabilization as the main effect in enhancing chemical rates and provide a unified view of ECD's role in catalysis from an atomistic perspective.cis-trans isomerization | Cyclophilin A | enzyme catalysis | enzyme dynamics | Kramers' rate theory E nzymes accelerate reaction rates by several orders of magnitude, allowing them to occur at timescales relevant for cellular functions (1). One of the long-standing issues in biochemistry is how enzymes achieve this remarkable speedup. It is commonly accepted that the most dominating effect arises from significant reduction in the free energy barrier compared to the corresponding noncatalyzed reaction in solution. It is also well established that this predominant effect is mainly electrostatic in nature (2, 3), which is more favorable for the transition state than the reactant or the product (1). However, to what degree and how other factors such as desolvation, steric strain, and enzyme dynamics contribute to catalysis remains disputable. Of particular interest is the role of enzyme dynamics in catalysis that has stirred considerable debate (4-11) partly because it has not been clearly defined, leading to a semantic issue. Also, the link between enzyme dynamics and catalysis is difficult to address both experimentally and theoretically. Currently, the implications of enzyme dynamics are from ensemble-and time-averaged experiments, as the temporal behavior of every atom cannot be observed directly. Although standard molecular dynamics (MD) simulations can provide an a...

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Section: Accelerating Cypa Dynamics and Its Effects On The Chemicalsupporting

confidence: 90%

Resolving the complex role of enzyme conformational dynamics in catalytic function

Doshi

McGowan

Ladani

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Together with the further development of spin relaxation techniques and isotope labeling strategies it is likely that protein dynamics will also be possible to study from chemical shifts. There has been development in this direction (Lehtivarjo et al 2012;Markwick et al 2010;Robustelli et al 2012) and the time averaging of chemical shifts in these methods is based on molecular dynamics simulations, which means that fast time scale motions can be addressed. As an example, Vendruscolo and co-workers developed a methodology that enabled characterization of a conformational equilibrium in Rnase A by including chemical shifts as restraints in molecular dynamics simulations (Camilloni et al 2012(Camilloni et al , 2013.…”

Section: Discussionmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

149

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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“…We investigated the conformational properties of cyclophilin A during the proline isomerization process by using NMR spectroscopy, which can provide atomic-resolution descriptions of the motions of macromolecules in solution (24)(25)(26)(27)(28)(29)(30)(31)(32). In our strategy, NMR data are used as replica-averaged structural restraints in molecular dynamics simulations.…”

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confidence: 99%

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

Camilloni

Sahakyan

Holliday

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.enzyme catalysis | NMR spectroscopy D ifferent families of enzymes, often referred to as "peptidylprolyl isomerases" (PPIases), catalyze proline isomerization, a process that involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline (1-3). This isomerization process is an intrinsically slow reaction, typically occurring on the time scale of several minutes under physiological conditions. Hence it often represents a ratelimiting step in biochemical reactions and indeed is used ubiquitously as a molecular switch in regulation (1-7).The possible mechanisms by which PPIases speed up this reaction have been the subject of intense scrutiny (8-16), although consensus descriptions of such mechanisms have not yet emerged. A question of particular relevance is the specific manner in which the electrostatic field in the catalytic site may facilitate the isomerization reaction. To investigate this problem, we considered the case of cyclophilin A, a member of the cyclophilin family of . Previous studies have suggested that conformations resembling those typical of the cis-bound and the trans-bound states are populated through conformational fluctuations in the free state of the enzyme and therefore functional insights into its mechanism of action might be obtained from the study of the free state (21-23).The approach that we followed in studying the mechanism of action of cyclophilin A is based on the simultaneous determination of the structures of the cis-bound and trans-bound states of the complex between the enzyme and its substrate as the catalytic process takes place. Our results re...

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Enhanced Conformational Space Sampling Improves the Prediction of Chemical Shifts in Proteins

Cited by 81 publications

References 16 publications

Resolving the complex role of enzyme conformational dynamics in catalytic function

Resolving the complex role of enzyme conformational dynamics in catalytic function

Protein dynamics and function from solution state NMR spectroscopy

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

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