NMR and protein dynamics

NMR techniques can give insight into a wide variety of motional events that occur in proteins over a range of timescales. In the first section of this article an overview of the results of dynamics studies, using NMR methods, on both small globular and larger multi-domain proteins is presented including the findings from investigations of non-native partly folded states. The second section of the article then concentrates on two topics where NMR can give residue specific quantitative data, namely coupling constant measurements and relaxation studies, including comparisons of these NMR data with results from crystallographic studies and theoretical molecular dynamics simulations. Finally the possible functional significance of the experimentally observed motions in proteins is discussed. © 1996 John Wiley & Sons, Inc.     

Interpreting protein structural dynamics from NMR chemical shifts

In this investigation, semiempirical NMR chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (MD) simulations of proteins. MD-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static X-ray structures. Improved chemical shift predictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations within conformational basins. Improved chemical shift predictions also result from discrete changes to conformations observed in X-ray structures, which may result from crystal contacts, and are not always reflective of conformational dynamics in solution. Chemical shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, and averaged (1)H chemical shifts are particularly sensitive reporters of fluctuations in aromatic ring positions and geometries of hydrogen bonds. In addition, poor predictions of MD-averaged chemical shifts can identify spurious conformations and motions observed in MD simulations that may result from force field deficiencies or insufficient sampling and can also suggest subsets of conformational space that are more consistent with experimental data. These results suggest that the analysis of dynamically averaged NMR chemical shifts from MD simulations can serve as a powerful approach for characterizing protein motions in atomistic detail.     

Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis

A novel iterative procedure is described that allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. If five orthogonal alignments are available, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in the previously well-studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous (15)N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than those found by (15)N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction.     

Role of protein flexibility in the design of Bcl-XL targeting agents: insight from molecular dynamics

Detailed understanding of protein–ligand interactions is crucial to the design of more effective drugs. This is particularly true when targets are protein interfaces which have flexible, shallow binding sites that exhibit substantial structural rearrangement upon ligand binding. In this study, we use molecular dynamics simulations and free energy calculations to explore the role of ligand-induced conformational changes in modulating the activity of three generations of Bcl-X_L inhibitors. We show that the improvement in the binding affinity of each successive ligand design is directly related to a unique and measurable reduction in local flexibility of specific regions of the binding groove, accompanied by the corresponding changes in the secondary structure of the protein. Dynamic analysis of ligand–protein interactions reveals that the latter evolve with each new design consistent with the observed increase in protein stability, and correlate well with the measured binding affinities. Moreover, our free energy calculations predict binding affinities which are in qualitative agreement with experiment, and indicate that hydrogen bonding to Asn100 could play a prominent role in stabilizing the bound conformations of latter generation ligands, which has not been recognized previously. Overall our results suggest that molecular dynamics simulations provide important information on the dynamics of ligand–protein interactions that can be useful in guiding the design of small-molecule inhibitors of protein interfaces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.     

Temperature dependence of NMR order parameters and protein dynamics

The helical subdomain, HP36, of the F-actin-binding headpiece domain of chicken villin, is the smallest naturally occurring polypeptide that folds to a thermostable compact structure. Unconstrained molecular dynamics simulations and constrained molecular dynamics simulations using umbrella sampling are used to study the temperature dependence of internal motions of the backbone amide moieties of HP36. The potential of mean force (PMF) for the N-H bond vector, determined from the constrained simulations, is found to be temperature dependent. A simple analytical expression is derived that describes the temperature dependence of the PMF. The parameters of this model are obtained from the PMF, from the unconstrained molecular dynamics simulations, or from experimental values of the generalized order parameter. The results provide a linkage between experimental and theoretical measures of the temperature dependence of protein motions.     

An NMR study of the solution dynamics of deltorphin-I

Nuclear spin relaxation rates associated with cross-correlated, dipole-dipole interactions are enlisted to help characterize the solution state dynamics of a small heptapeptide, deltorphin-I. A simple two-site jump model can be used to interpret the data obtained on two specific (13)C labeled residues, D-alanine and glycine. The influence of temperature and solvent upon the observed dynamics is investigated. Similarly, relaxation rates associated with dipole-shielding anisotropy interferences are used to examine the magnitude and orientation of various chemical shielding tensors within the D-alanine and glycine residues.     

2H NMR studies of glycerol dynamics in protein matrices

We use (2)H NMR spectroscopy to investigate the rotational motion of glycerol molecules in matrices provided by the connective tissue proteins elastin and collagen. Analyzing spin-lattice relaxation, line-shape properties, and stimulated-echo decays, we determine the rates and geometries of the motion as a function of temperature and composition. It is found that embedding glycerol in an elastin matrix leads to a mild slowdown of glycerol reorientation at low temperatures and glycerol concentrations, while the effect vanishes at ambient temperatures or high solvent content. Furthermore, it is observed that the nonexponential character of the rotational correlation functions is much more prominent in the elastin matrix than in the bulk liquid. Results from spin-lattice relaxation and line shape measurements indicate that, in the mixed systems, the strong nonexponentiality is in large part due to the existence of distributions of correlation times, which are broader on the long-time flank and, hence, more symmetric than in the neat system. Stimulated-echo analysis of slow glycerol dynamics reveals that, when elastin is added, the mechanism for the reorientation crosses over from small-angle jump dynamics to large-angle jump dynamics and the geometry of the motion changes from isotropic to anisotropic. The results are discussed against the background of present and previous findings for glycerol and water dynamics in various protein matrices and compared with observations for other dynamically highly asymmetric mixtures so as to ascertain in which way the viscous freezing of a fast component in the matrix of a slow component differs from the glassy slowdown in neat supercooled liquids.     

Protein dynamics from a NMR perspective: networks of coupled rotators and fractional Brownian dynamics

Nuclear magnetic resonance (NMR) has proven to be the most valuable tool for investigating internal dynamics of proteins. In this perspective, the interpretation of NMR relaxation data eventually relies on a model of the motions. In this article, we propose to compare two radically different approaches that aim at describing internal dynamics in proteins. It is shown that the correlation functions predicted by a network of coupled rotators can be interpreted in terms of a heuristic approach based on fractional Brownian dynamics for each of the vectors in the network. Our results are interpreted in terms of the probability distributions of relaxation modes in both processes, the median of which turns out to be the relevant quantity for the comparison of both models.     

Enzymatic GTP hydrolysis: insights from an ab initio molecular dynamics study

Ab initio methods were used to shed light on fundamental aspects of the enzymatic mechanism of guanosine triphosphate hydrolysis in the Cdc42/Cdc42GAP complex. The calculations focused on the nucleophilic addition of the catalytic water molecule to the gamma-phosphate phosphorus atom. A large model system was required to correctly reproduce the electrostatic properties on the active site. The model turned out to reproduce most of the electrostatic field of the biological complex at the reactants. Our calculations established the H-bond pattern of the catalytic water (WAT), which turned out to interact with Q61 and T35, in the most stable conformation. This ruled out the possibility that the catalytic water transferred its proton directly to the gamma-phosphate. Furthermore, the calculations suggested that the electronic structure of WAT was very different from that in the bulk. Finally, this study showed that during the reaction, WAT transferred a proton to Gln61, consistent with the available X-ray data on a transition-state analogue/enzyme complex(19) and with the decrease of activity in the Q61E mutant.     

Structural features of carbon products: an NMR study

Structural characteristics of a series of carbon products and intermediates of carbonization of some polymers were studied by high-resolution solid-state ¹³C NMR spectroscopy, with comparison of the experimental and calculated spectroscopic data.     

Application of principal component analysis in protein unfolding: an all-atom molecular dynamics simulation study

We have performed molecular dynamics (MD) simulation of the thermal denaturation of one protein and one peptide-ubiquitin and melittin. To identify the correlation in dynamics among various secondary structural fragments and also the individual contribution of different residues towards thermal unfolding, principal component analysis method was applied in order to give a new insight to protein dynamics by analyzing the contribution of coefficients of principal components. The cross-correlation matrix obtained from MD simulation trajectory provided important information regarding the anisotropy of backbone dynamics that leads to unfolding. Unfolding of ubiquitin was found to be a three-state process, while that of melittin, though smaller and mostly helical, is more complicated.     

Spin dynamics in the modulation frame: application to homonuclear recoupling in magic angle spinning solid-state NMR

We introduce a family of solid-state NMR pulse sequences that generalizes the concept of second averaging in the modulation frame and therefore provides a new approach to perform magic angle spinning dipolar recoupling experiments. Here, we focus on two particular recoupling mechanisms-cosine modulated rotary resonance (CMpRR) and cosine modulated recoupling with isotropic chemical shift reintroduction (COMICS). The first technique, CMpRR, is based on a cosine modulation of the rf phase and yields broadband double-quantum (DQ) (13)C recoupling using >70 kHz omega(1,C)/2pi rf field for the spinning frequency omega(r)/2=10-30 kHz and (1)H Larmor frequency omega(0,H)/2pi up to 900 MHz. Importantly, for p>or=5, CMpRR recouples efficiently in the absence of (1)H decoupling. Extension to lower p values (3.5相似文献   

Alignment of lamellar diblock copolymer phases under shear: insight from dissipative particle dynamics simulations

Sheared self-assembled lamellar phases formed by symmetrical diblock copolymers are investigated through dissipative particle dynamics simulations. Our intent is to provide insight into the experimental observations that the lamellar phases adopt parallel alignment at low shear rates and perpendicular alignment at high shear rates and that it is possible to use shear to induce a transition from the parallel to perpendicular alignment. Simulations are initiated either from lamellar structures prepared under zero shear where lamellae are aligned into parallel, perpendicular, or transverse orientations with respect to the shear direction or from a disordered melt obtained by energy minimization of a random structure. We first consider the relative stability of the parallel and perpendicular phases by applying shear to lamellar structures initially aligned parallel and perpendicular to the shear direction, respectively. The perpendicular lamellar phase persists for all shear rates investigated, whereas the parallel lamellar phase is only stable at low shear rates, and it becomes unstable at high shear rates. At the high shear rates, the parallel lamellar phase first transforms into an unstable diagonal lamellar phase; and upon further increase of the shear rate, the parallel lamellar phase reorients into a perpendicular alignment. We further determine the preferential alignment of the lamellar phases at low shear rate by performing the simulations starting from either the initial transverse lamellar structure or the disordered melt. Since the low shear-rate simulations are plagued by the unstable diagonal lamellar phases, we vary the system size to achieve the natural spacing of the lamellae in the simulation box. In such cases, the unstable diagonal lamellar phases disappear and lamellar phases adopt the preferential alignment, either parallel or perpendicular. In agreement with the experimental observations, the simulations show that the lamellar phase preferentially adopts the parallel orientation at low shear rates and the perpendicular orientation at high shear rates. The simulations further reveal that the perpendicular lamellar phase has lower internal energy than the parallel lamellar phase, whereas the entropy production of the perpendicular lamellar phase is higher with respect to the parallel lamellar phase. Values of the internal energy and entropy production for the unstable diagonal lamellar phases lie between the corresponding values for the parallel and perpendicular lamellar phases. These simulation results suggest that the relative stability of the parallel and perpendicular lamellar phases at low shear rates is a result of the interplay between competing driving forces in the system: (a) the system's drive to adopt a structure with the lowest internal energy and (b) the system's drive to stay in a stationary nonequilibrium state with the lowest entropy production.     

Excited state dynamics of liquid water: insight from the dissociation reaction following two-photon excitation

The authors use transient absorption spectroscopy to monitor the ionization and dissociation products following two-photon excitation of pure liquid water. The primary decay mechanism changes from dissociation at an excitation energy of 8.3 eV to ionization at 12.4 eV. The two channels occur with similar yield for an excitation energy of 9.3 eV. For the lowest excitation energy, the transient absorption at 267 nm probes the geminate recombination kinetics of the H and OH fragments, providing a window on the dissociation dynamics. Modeling the OH geminate recombination indicates that the dissociating H atoms have enough kinetic energy to escape the solvent cage and one or two additional solvent shells. The average initial separation of H and OH fragments is 0.7+/-0.2 nm. Our observation suggests that the hydrogen bonding environment does not prevent direct dissociation of an O-H bond in the excited state. We discuss the implications of our measurement for the excited state dynamics of liquid water and explore the role of those dynamics in the ionization mechanism at low excitation energies.     

Theoretical insight into the aggregation induced emission phenomena of diphenyldibenzofulvene: a nonadiabatic molecular dynamics study

The diphenyldibenzofulvene (DPDBF) molecule appears in two forms: ring open and ring closed. The former fluoresces weakly in solution, but it becomes strongly emissive in the solid phase, exhibiting an exotic aggregation-induced emission phenomenon. The latter presents a normal aggregation quenching phenomenon, as is expected. We implement nonadiabatic molecular dynamics based on the combination of time-dependent Kohn-Sham (TDKS) and density functional tight binding (DFTB) methods with Tully's fewest switches surface hopping algorithm to investigate the excited state nonradiative decay processes. From the analysis of the nonadiabatic coupling vectors, it is found that the low frequency twisting motion in the ring open DPDBF couples strongly with the electronic excitation and dissipates the energy efficiently. While in the closed form, such motion is blocked by a chemical bond. This leads to the nonradiative decay rate for the open form (1.4 ps) becoming much faster than the closed form (24.5 ps). It is expected that, in the solid state, the low frequency motion of the open form will be hindered and the energy dissipation pathway by nonradiative decay will be slowed, presenting a remarkable aggregation enhanced emission phenomenon.     

New insight into relaxation dynamics of an epoxy/hydroxy functionalized polybutadiene from dielectric and mechanical spectroscopy studies

Dielectric and mechanical spectroscopy methods have been employed to describe the temperature dependencies of the segmental and macromolecular relaxation rates in epoxy/hydroxy functionalized polybutadiene. Dielectric studies on the dynamics of segments of the polymer as well as the mobility of small ions trapped in the system have been carried out both as a function of temperature and pressure under isobaric and isothermal conditions, respectively.     

Structure and dynamics of the homologous series of alanine peptides: a joint molecular dynamics/NMR study

The phi,psi backbone angle distribution of small homopolymeric model peptides is investigated by a joint molecular dynamics (MD) simulation and heteronuclear NMR study. Combining the accuracy of the measured scalar coupling constants and the atomistic detail of the all-atom MD simulations with explicit solvent, the thermal populations of the peptide conformational states are determined with an uncertainty of <5 %. Trialanine samples mainly ( approximately 90%) a poly-l-proline II helix-like structure, some ( approximately 10%) beta extended structure, but no alphaR helical conformations. No significant change in the distribution of conformers is observed with increasing chain length (Ala(3) to Ala(7)). Trivaline samples all three major conformations significantly. Triglycine samples the four corner regions of the Ramachandran space and exists in a slow conformational equilibrium between the cis and trans conformation of peptide bonds. The backbone angle distribution was also studied for the segment Ala3 surrounded by either three or eight amino acids on both N- and C-termini from a sequence derived from the protein hen egg white lysozyme. While the conformational distribution of the central three alanine residues in the 9mer is similar to that for the small peptides Ala(3)-Ala(7), major differences are found for the 19mer, which significantly (30-40%) samples alphaR helical stuctures.