Life is marked by change over time, and biologists explore this phenomenon by watching, for example, Caenorhabditis elegans developing from embryos into adults, mice running in a cage, and nerve cells firing. In search of how and why, biology arrived at the molecular level. Understanding protein function on an atomic level has been revolutionized by high-resolution X-ray crystallography, resulting in a surge in studies of structure-function relationships. The detail in these colourful structures flooding the covers of modern journals can be deceptive, suggesting that one unique structure, the 'folded state' , is the final answer. Ironically, the dynamic nature of biology seems to have been forgotten at this microscopic level.Physicists, however, will object to a static picture: they see proteins as soft materials that sample a large ensemble of conformations around the average structure as a result of thermal energy. A complete description of proteins requires a multidimensional energy landscape that defines the relative probabilities of the conformational states (thermodynamics) and the energy barriers between them (kinetics). In biology, this concept has recently gained traction, leading to an extension of the structure-function paradigm to include dynamics. To understand proteins in action, the fourth dimension, time, must be added to the snapshots of proteins frozen in crystal structures. A major obstacle is that it is not possible to watch experimentally individual atoms moving within a protein. Instead, sophisticated biophysical methods are needed to measure the physical properties from which the dynamics can be inferred.In this review, we discuss how protein function is rooted in the energy landscape. The basic concepts and the biophysical methods are illustrated by several examples. To avoid past semantic confusion about the term protein dynamics, we define it as any time-dependent change in atomic coordinates. Protein dynamics thus includes both equilibrium fluctuations and non-equilibrium effects. The fluctuations observed at equilibrium seem to govern biological function in processes both near and far from equilibrium; therefore, we focus on these motions. Non-equilibrium effects are also called dynamical effects 1 (the source of confusion 2 ), and they have a minimal effect on the overall rates of biological processes 3,4 . Biological motors that convert chemical energy to mechanical energy 5,6 are not discussed here. The energy landscapeAlthough the idea of an energy landscape might be most familiar in the context of protein folding (for example, the folding funnel hypothesis) [7][8][9] , this concept had already been applied to folded proteins more than 30 years ago by Frauenfelder and co-workers . Subsequent studies on myoglobin led to the idea that substates are in thermal equilibrium and that both solvent 13-15 and ligands influence the landscape (Fig. 1a). At the glass transition temperature 10,12 , an increase in anharmonic dynamics occurs in proteins, and this is interpreted as the protein no...

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.

The synergy between structure and dynamics is essential to the function of biological macromolecules. Thermally driven dynamics on different timescales have been experimentally observed or simulated, and a direct link between micro- to milli-second domain motions and enzymatic function has been established. However, very little is understood about the connection of these functionally relevant, collective movements with local atomic fluctuations, which are much faster. Here we show that pico- to nano-second timescale atomic fluctuations in hinge regions of adenylate kinase facilitate the large-scale, slower lid motions that produce a catalytically competent state. The fast, local mobilities differ between a mesophilic and hyperthermophilic adenylate kinase, but are strikingly similar at temperatures at which enzymatic activity and free energy of folding are matched. The connection between different timescales and the corresponding amplitudes of motions in adenylate kinase and their linkage to catalytic function is likely to be a general characteristic of protein energy landscapes.

The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.

Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory.

A longstanding challenge is to understand at the atomic level how protein dynamics contribute to enzyme catalysis. X-ray crystallography can provide snapshots of conformational substates sampled during enzymatic reactions1, while NMR relaxation methods reveal the rates of interconversion between substates and the corresponding relative populations1,2. However, these current methods cannot simultaneously reveal the detailed atomic structures of the rare states and rationalize the finding that intrinsic motions in the free enzyme occur on a time scale similar to the catalytic turnover rate. Here we introduce dual strategies of ambient-temperature X-ray crystallographic data collection and automated electron-density sampling to structurally unravel interconverting substates of the human proline isomerase, cyclophilin A (CypA). A conservative mutation outside the active site was designed to stabilize features of the previously hidden minor conformation. This mutation not only inverts the equilibrium between the substates, but also causes large, parallel reductions in the conformational interconversion rates and the catalytic rate. These studies introduce crystallographic approaches to define functional minor protein conformations and, in combination with NMR analysis of the enzyme dynamics in solution, show how collective motions directly contribute to the catalytic power of an enzyme.

A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.

Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.