Intrinsic dynamics of an enzyme underlies catalysis
TL;DR: It is shown that the intrinsic plasticity of the protein is a key characteristic of catalysis, and the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergy pressure between structure and dynamics.
Abstract: A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
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TL;DR: The dream is to 'watch' proteins in action in real time at atomic resolution, which requires addition of a fourth dimension, time, to structural biology so that the positions in space and time of all atoms in a protein can be described in detail.
Abstract: Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromolecules execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is to 'watch' proteins in action in real time at atomic resolution. This requires addition of a fourth dimension, time, to structural biology so that the positions in space and time of all atoms in a protein can be described in detail.
2,109 citations
TL;DR: Experimental evidence supports a new molecular recognition paradigm for processes as diverse as signaling, catalysis, gene regulation and protein aggregation in disease, which has the potential to significantly impact views and strategies in drug design, biomolecular engineering and molecular evolution.
Abstract: Molecular recognition is central to all biological processes. For the past 50 years, Koshland's 'induced fit' hypothesis has been the textbook explanation for molecular recognition events. However, recent experimental evidence supports an alternative mechanism. 'Conformational selection' postulates that all protein conformations pre-exist, and the ligand selects the most favored conformation. Following binding the ensemble undergoes a population shift, redistributing the conformational states. Both conformational selection and induced fit appear to play roles. Following binding by a primary conformational selection event, optimization of side chain and backbone interactions is likely to proceed by an induced fit mechanism. Conformational selection has been observed for protein-ligand, protein-protein, protein-DNA, protein-RNA and RNA-ligand interactions. These data support a new molecular recognition paradigm for processes as diverse as signaling, catalysis, gene regulation and protein aggregation in disease, which has the potential to significantly impact our views and strategies in drug design, biomolecular engineering and molecular evolution.
1,699 citations
TL;DR: An upper bound for the approximation error made by modeling molecular dynamics with a Markov chain is described and it is shown that this error can be made arbitrarily small with surprisingly little effort.
Abstract: Markov state models of molecular kinetics (MSMs), in which the long-time statistical dynamics of a molecule is approximated by a Markov chain on a discrete partition of configuration space, have seen widespread use in recent years. This approach has many appealing characteristics compared to straightforward molecular dynamics simulation and analysis, including the potential to mitigate the sampling problem by extracting long-time kinetic information from short trajectories and the ability to straightforwardly calculate expectation values and statistical uncertainties of various stationary and dynamical molecular observables. In this paper, we summarize the current state of the art in generation and validation of MSMs and give some important new results. We describe an upper bound for the approximation error made by modeling molecular dynamics with a MSM and we show that this error can be made arbitrarily small with surprisingly little effort. In contrast to previous practice, it becomes clear that the best MSM is not obtained by the most metastable discretization, but the MSM can be much improved if non-metastable states are introduced near the transition states. Moreover, we show that it is not necessary to resolve all slow processes by the state space partitioning, but individual dynamical processes of interest can be resolved separately. We also present an efficient estimator for reversible transition matrices and a robust test to validate that a MSM reproduces the kinetics of the molecular dynamics data.
1,082 citations
Cites background from "Intrinsic dynamics of an enzyme und..."
...With the ever increasing time resolution of ensemble kinetics experiments and the more recent maturation of sensitive single-molecule techniques in biophysics, experimental evidence supporting the near-universality of the existence of multiple metastable conformational substates and complex kinetics in biomolecules has continued to accumulate [7, 8, 9, 10, 11, 12, 13]....
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1,075 citations
TL;DR: A structural ensemble of ubiquitin, refined against residual dipolar couplings, comprising solution dynamics up to microseconds, covers the complete structural heterogeneity observed in 46 ubiquitIn crystal structures, most of which are complexes with other proteins.
Abstract: Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.
1,000 citations
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01 Jan 1969
2,160 citations
"Intrinsic dynamics of an enzyme und..." refers background or methods or result in this paper
...Chemical shifts of the minor enzyme form (qEminor) were calculated by using Dq obtained from the dispersion experiments (equation (1)) together with the sign of Dq determined from the HMQC/HSQC experiment....
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...type CypA because larger populations would yield Dq values (equation (1)) that were so small as to result in unobservable chemical-shift changes in (15)N HSQC experiments for the mutants....
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...It is also noteworthy that the calculated chemical-shift differences (equation (1)) between Emajor and Eminor (qEmajor 2 qEminor; scheme (3)) are similar to those between EScis and EStrans (qEScis 2 qEStrans; scheme (2)) for residues remote from the substrate, suggesting that the cis substrate binds Emajor to form EScis, whereas the trans substrate binds Eminor to form EStrans....
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...This must reflect a shift in relative populations of the two exchanging states because kex remains the same (equation (1))....
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TL;DR: An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships.
1,862 citations
TL;DR: The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies, similar to how myoglobin achieves specificity and order.
Abstract: Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.
1,448 citations
TL;DR: A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion that have important implications for the origin and evolution of enzymes, as well as for protein engineering.
Abstract: The seminal hypotheses proposed over the years for enzymatic catalysis are scrutinized. The historical record is explored from both biochemical and theoretical perspectives. Particular attention is given to the impact of molecular motions within the protein on the enzyme's catalytic properties. A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion. Such coupled networks have important implications for the origin and evolution of enzymes, as well as for protein engineering.
1,068 citations
"Intrinsic dynamics of an enzyme und..." refers background or result in this paper
...It also implies that the chemical shifts between the two states (Dq),and hence their structural differences, are identical for wild-type CypA and all CypA mutants (scheme (3)); Supplementary Table S2)....
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...It is also noteworthy that the calculated chemical-shift differences (equation (1)) between Emajor and Eminor (qEmajor 2 qEminor; scheme (3)) are similar to those between EScis and EStrans (qEScis 2 qEStrans; scheme (2)) for residues remote from the substrate, suggesting that the cis substrate binds Emajor to form EScis, whereas the trans substrate binds Eminor to form EStrans....
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TL;DR: A framework for understanding the effects of lowering of the activation free energy and changes in the generalized transmission coefficient on enzyme catalysis is presented, and the contributions of the different factors are identified and quantified by computer simulations.
Abstract: Advances in transition state theory and computer simulations are providing new insights into the sources of enzyme catalysis. Both lowering of the activation free energy and changes in the generalized transmission coefficient (recrossing of the transition state, tunneling, and nonequilibrium contributions) can play a role. A framework for understanding these effects is presented, and the contributions of the different factors, as illustrated by specific enzymes, are identified and quantified by computer simulations. The resulting understanding of enzyme catalysis is used to comment on alternative proposals of how enzymes work.
1,003 citations