Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

Structural biology by NMR: structure, dynamics, and interactions

The function of bio-macromolecules is determined by both their 3D structure and conformational dynamics. These molecules are inherently flexible systems displaying a broad range of dynamics on time-scales from picoseconds to seconds. Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as the method of choice for studying both protein structure and dynamics in solution. Typically, NMR experiments are sensitive both to structural features and to dynamics, and hence the measured data contain information on both. Despite major progress in both experimental approaches and computational methods, obtaining a consistent view of structure and dynamics from experimental NMR data remains a challenge. Molecular dynamics simulations have emerged as an indispensable tool in the analysis of NMR data.     

Application of NMR and EPR methods to the study of RNA

The application of techniques based on magnetic resonance, specifically electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR), has provided a wealth of new information on RNA structures, as well as insights into the dynamics and function of these important biomolecules. NMR spectroscopy is very successful for determining the solution structures of small RNA domains, aptamers and ribozymes, and exploring their intramolecular dynamics and interactions with ligands. EPR-based methods have been used to map local dynamic and structural features of RNA, to explore different modes of RNA-ligand interaction, to obtain long-range structural restraints and to probe metal-ion-binding sites.     

Dynamic activation of protein function: a view emerging from NMR spectroscopy.

Recent developments in solution NMR methods have allowed for an unprecedented view of protein dynamics. Current insights into the nature of protein dynamics and their potential influence on protein structure, stability and function are reviewed. Particular emphasis is placed on the potential of fast side chain motion to report on the residual conformational entropy of proteins and how this entropy can enter into both the thermodynamic and kinetic aspects of protein function.     

Oligomeric structure, dynamics, and orientation of membrane proteins from solid-state NMR

Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both alpha-helical and beta sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information.     

An Introduction to Biological NMR Spectroscopy

NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP).     

Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes

Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys⁴⁰, residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail.     

The dynamic duo: Combining NMR and small angle scattering in structural biology

Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X‐ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well‐suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR‐derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state‐of‐the‐art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution.     

Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on (15)N and (2)H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in μs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect.     

Comparative proton nuclear magnetic resonance in cancerous and healthy tissues of mice, with variable frequency and temperature

We have measured the proton longitudinal relaxation time T1 in healthy tissues and tumors as a function of the Larmor frequency and temperature. We deduce the proportions and dynamics of bound and free water as differenciation criteria for NMR imaging.     

Phosphorus-31 NMR as a probe for phosphoproteins

Nuclear magnetic resonance methodology continues to advance such that phosphorus-31 NMR experiments can be profitably applied to elucidate some aspects of proteins which are covalently phosphorylated. This review introduces NMR spectral parameters pertinent to using phosphorus-31 NMR for investigation of structure and dynamics. The techniques of two-dimensional NMR, solid state NMR, and isotopic substitution are also introduced. Characteristics of phosphorylated amino acids and peptides, as revealed by phosphorus-31 NMR, are described. Studies of phosphorylated containing phosphomonoesters, phosphoramidates, acyl phosphates, and disubstituted phosphorus bridges are discussed. Among these phosphoproteins are several examples where phosphorus residues evidently play a role as polyelectrolytes, in enzyme catalysis, and in regulation of protein function.     

The dynamics,structure, and conformational free energy of proline-containing antifreeze glycoprotein

Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed.     

Structure refinement of a cyclic peptide from two-dimensional NMR data and molecular modeling

The conformational and dynamic properties of a cyclic peptide designed to inhibit human renin have been examined by using NMR and molecular modeling. From a quantitative analysis of a series of two-dimensional NOE data sets, proton-proton distances were calculated. Several different methods were explored and compared to incorporate these distance constraints as well as those derived from vicinal spin-spin coupling constants into computer-generated three-dimensional structures. These methods included interactive manual manipulation of the structures to fit the NMR-determined distance constraints, distance geometry, constrained energy minimizations, and constrained molecular dynamics. The advantages and disadvantages of the methods are discussed. In addition, to gain insight into the conformations accessible to the cyclic peptide and the relative flexibility of the different parts of the molecule, molecular dynamics calculations were performed at three different temperatures. Average interproton distances and dihedral angles were obtained from the structures generated in the dynamics trajectories and compared to those obtained from the NMR experiments. Despite the four methylene groups and ether linkage contained in the cyclic portion of the peptide, our NMR results indicated a preferred conformation for the macrocyclic ring of the peptide and supported the presence of a cis Phe-Ala peptide bond. In contrast, both the molecular dynamics and NMR data indicated a considerable amount of flexibility for the remaining noncyclic portion of the molecule. These results are used to propose an explanation for the cyclic peptide's inability to inhibit human renin.     

Probing conformational dynamics in biomolecules via chemical exchange saturation transfer: a primer

Although Chemical Exchange Saturation Transfer (CEST) type NMR experiments have been used to study chemical exchange processes in molecules since the early 1960s, there has been renewed interest in the past several years in using this approach to study biomolecular conformational dynamics. The methodology is particularly powerful for the study of sparsely populated, transiently formed conformers that are recalcitrant to investigation using traditional biophysical tools, and it is complementary to relaxation dispersion and magnetization transfer experiments that have traditionally been used to study chemical exchange processes. Here we discuss the concepts behind the CEST experiment, focusing on practical aspects as well, we review some of the pulse sequences that have been developed to characterize protein and RNA conformational dynamics, and we discuss a number of examples where the CEST methodology has provided important insights into the role of dynamics in biomolecular function.     

NMR reveals novel mechanisms of protein activity regulation

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.     

A solution NMR view of protein dynamics in the biological membrane

Structure determination of membrane-associated proteins (MPs) represents a frontier of structural biology that is characterized by unique challenges in sample preparation and data acquisition. No less important is our ability to study the dynamics of MPs, since MP flexibility and characteristic motions often make sizeable contributions to their function. This review focuses on solution state NMR methods to characterize dynamics of MPs in the membrane environment. NMR approaches to study molecular motions on a wide range of time-scales and their application to membrane proteins are described. Studies of polytopic and bitopic MPs demonstrating the power of such methods to characterize the dynamic behavior of MPs and their interaction with the membrane-mimicking surroundings are presented. Attempts are made to place the dynamic conclusions into a biological context. The importance and limitations of such investigations guarantee that further developments in this field will be actively pursued.     

Molecular mechanics and dynamics of an abasic frameshift in DNA and comparison to NMR data

In a previous publication (Ph. Cuniasse, L.C. Sowers, R. Eritja, B. Kaplan, M.F. Goodman, J.A.H. Cognet, M. Le Bret, W. Guschlbauer and G.V. Fazakerley, Biochemistry 28, 2018 (1989), we determined by two dimensional NMR studies and molecular mechanics calculations the three-dimensional structure of a non-selfcomplementary oligonucleotide: [sequence; see text] where dr, at the center of the first strand, is a model abasic site. In order to explain all the results arising from NMR measurements, we found that an equilibrium between two conformations was necessary. These conformations differ mainly by the sugar pucker of G5 which is C2' endo or C3' endo. The latter is stabilized by addition of counterions between phosphate residues P3 and P4. In this paper, we have constructed systematically, all possible structures as a function of torsion angles delta of dr4 and of G5 by molecular mechanics in the presence or absence of counterions. Since these conformations were not forced with NMR distance measurements, this method allows detailed comparisons between all possible conformations and NMR data. Maps of contour lines of the potential energy, of fits to NMR distance measurements, and of helical twist as a function of torsion angles delta of dr4 and of G5 unravel the difficulties associated with the study of the G5 sugar pucker conformation equilibrium. Sugar puckers and proton distances are very sensitive criteria to monitor molecular dynamics. Relying on these experimental criteria, we have tested many molecular dynamics preparation phases and we propose a new warm-up and equilibration procedure for molecular dynamics. Thus we show with a 290 ps molecular dynamic run that G5 is in conformational equilibrium and that all NMR data are well reproduced.     

NMR insights into dynamics regulated target binding of DLC8 dimer

Conformational dynamics play a crucial role in biological function. Dynein light chain protein (DLC8) acts as a cargo adaptor, and exists as a dimer under physiological conditions and dissociates into monomer below pH 4. In the present NMR study, we identified some dynamic residues in the dimer using chemical shift perturbation approach by applying small pH change. As evidenced by gel filtration and CD studies, this small pH change does not alter the globular structural features of the protein. In fact, these changes result in small local stability perturbations as monitored using temperature dependence of amide proton chemical shifts, and influence the dynamics of the dimer substantially. Further, interaction studies of the protein with a peptide containing the recognition motif of cargo indicated that the efficacy of peptide binding decreases when the pH is reduced from 7 to 6. These observations taken together support the conception that dynamics can regulate cargo binding/trafficking by the DLC8 dimer.     

On using time-averaging restraints in molecular dynamics simulation

Introducing experimental values as restraints into molecular dynamics (MD) simulations to bias the values of particular molecular properties, such as nuclear Overhauser effect intensities or distances, 3J coupling constants, chemical shifts or crystallographic structure factors, towards experimental values is a widely used structure refinement method. To account for the averaging of experimentally derived quantities inherent in the experimental techniques, time-averaging restraining methods may be used. In the case of structure refinement using 3J coupling constants from NMR experiments, time-averaging methods previously proposed can suffer from large artificially induced structural fluctuations. A modified time-averaged restraining potential energy function is proposed which overcomes this problem. The different possible approaches are compared using stochastic dynamics simulations of antamanide, a cyclic peptide of ten residues.