Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

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202

The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure-function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.energy landscape | transiently and sparsely populated biomolecular states | invisible states | structure-function paradigmThe field of structural biology emerged from seminal X-ray diffraction studies of biomolecules such as DNA (1), myoglobin (2), hemoglobin (3), and lysozyme (4) that were carried out over 50 years ago. Since these landmark investigations, our knowledge of the relation between structure and function has greatly expanded, driven by significant improvements in both experimental and computational approaches and, of course, by the exponential increase in the number of structures that are now available. Despite tremendous advances, structural biology remains largely focused on studies of the lowest-energy conformational states of biomolecules, and although there are notable exceptions (5), the end game often remains the determination of a single static 3D structure that is then used as a starting point for understanding molecular function. It is becoming increasingly clear, however, that this narrow approach is not sufficient and that a description of molecular structure must take into account conformational fluctuations that can occur over a broad range of both time and length scales.The conformational space that can be explored by a given biomolecule is usually explained by invoking the concept of an energy landscape (6), a multidimensional hypersurface that governs the thermodynamics and kinetics of conformational transitions (7,8). Because biomolecular stability is dictated by the sum of a large number of attractive and repulsive interactions of similar strength, the resultant free-energy landscape is rugged (9), with the global minimum in the surface thought to correspond to the native state of the biomolecule. In addition, there are often local minima that are separated from the global minimum by free-energy barriers that can be overcome by thermal fluctuations. These low-lying conformationally excited states have remained elusive to quantitative investigation because their sparse population and transient nature complicates their study by conventional structural biology techniques that are so powerfu...

Section: Applications To Other Transient Conformersmentioning

confidence: 99%

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers

Sekhar

Kay

2013

235

202

“…enzyme dynamics | hydrogen tunneling | path integral | ring polymer molecular dynamics P rotein motions are central to enzyme catalysis, with conformational changes on the micro-and millisecond timescale wellestablished to govern progress along the catalytic cycle (1,2). Less is known about the role of faster, atomic-scale fluctuations that occur in the protein environment of the active site.…”

mentioning

confidence: 99%

Dynamics and dissipation in enzyme catalysis

Boekelheide

Salomón-Ferrer²,

Miller³

2011

153

160

We use quantized molecular dynamics simulations to characterize the role of enzyme vibrations in facilitating dihydrofolate reductase hydride transfer. By sampling the full ensemble of reactive trajectories, we are able to quantify and distinguish between statistical and dynamical correlations in the enzyme motion. We demonstrate the existence of nonequilibrium dynamical coupling between protein residues and the hydride tunneling reaction, and we characterize the spatial and temporal extent of these dynamical effects. Unlike statistical correlations, which give rise to nanometer-scale coupling between distal protein residues and the intrinsic reaction, dynamical correlations vanish at distances beyond 4-6 Å from the transferring hydride. This work finds a minimal role for nonlocal vibrational dynamics in enzyme catalysis, and it supports a model in which nanometer-scale protein fluctuations statistically modulate-or gate-the barrier for the intrinsic reaction.enzyme dynamics | hydrogen tunneling | path integral | ring polymer molecular dynamics P rotein motions are central to enzyme catalysis, with conformational changes on the micro-and millisecond timescale wellestablished to govern progress along the catalytic cycle (1, 2). Less is known about the role of faster, atomic-scale fluctuations that occur in the protein environment of the active site. The textbook view of enzyme-catalyzed reaction mechanisms neglects the functional role of such fluctuations and describes a static protein environment that both scaffolds the active-site region and reduces the reaction barrier (3). This view has grown controversial amid evidence that active-site chemistry is coupled to motions in the enzyme (4-6), and it has been explicitly challenged by recent proposals that enzyme-catalyzed reactions are driven by vibrational excitations that channel energy into the intrinsic reaction coordinate (7,8) or promote reactive tunneling (9, 10). In the following, we combine quantized molecular dynamics and rareevent sampling methods to determine the mechanism by which protein motions couple to reactive tunneling in dihydrofolate reductase and to clarify the role of nonequilibrium vibrational dynamics in enzyme catalysis.Manifestations of enzyme motion include both statistical and dynamical correlations. Statistical correlations are properties of the equilibrium ensemble and describe, for example, the degree to which fluctuations in the spatial position of one atom are influenced by fluctuations in another; these correlations govern the free-energy (FE) landscape and determine the transition state theory kinetics of the system (6). Dynamical correlations are properties of the time-evolution of the system and describe coupling between inertial atomic motions, as in a collective vibrational mode. Compelling evidence for long-ranged (i.e., nanometer-scale) networks of statistical correlations in enzymes emerges from genomic analysis (11), molecular dynamics simulations (11-13), and kinetic studies of double-mutant enzymes (14-16). But the rol...

“…A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2)(3)(4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5)(6)(7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2-4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5-11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12-21).A case in point is the nitric oxide synthase (NOS) enzymes (22-24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached "oxygenase" domain, which then enables NO synthesis to begin (22,(25)(26)(27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly...