Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.     

Probing protein side chain dynamics via 13C NMR relaxation

Protein side chain dynamics is associated with protein stability, folding, and intermolecular interactions. Detailed dynamics information is crucial for the understanding of protein function and biochemical and biophysical properties, which can be obtained using NMR relaxation techniques. In this review, (13)C relaxation of methine, methylene and methyl groups with and without (1)H decoupling are described briefly for a better understanding of how spin relaxation is associated with motional (dynamics) parameters. Developments in the measurement and interpretation of (13)C auto-relaxation and cross-correlated relaxation data are presented too. Finally, recent progress in the use of (13)C relaxation to probe the dynamics of protein side chains is detailed mainly for the dynamics of non-deuterated proteins on picoseconds-nanosecond timescales.     

Main chain and side chain dynamics of a heme protein: 15N and 2H NMR relaxation studies of R. capsulatus ferrocytochrome c2

A detailed characterization of the main chain and side chain dynamics in R. capsulatus ferrocytochrome c(2) derived from (2)H NMR relaxation of methyl group resonances is presented. (15)N relaxation measurements confirm earlier results indicating that R. capsulatus ferrocytochrome c(2) exhibits minor rotational anisotropy in solution. The current study is focused on the use of deuterium relaxation in side chain methyl groups, which has been shown to provide a detailed and accurate measure of internal dynamics. Results obtained indicate that the side chains of ferrocytochrome c(2) exhibit a wide range of motional amplitudes, but are more rigid than generally found in the interior of nonprosthetic group bearing globular proteins. This unusual rigidity is ascribed to the interactions of the protein with the large heme prosthetic group. This observation has significant implications for the potential of the heme-protein interface to modulate the redox properties of the protein and also points to the need for great precision in the design and engineering of heme proteins.     

Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena.

Oxidized flavodoxin from Cyanobacterium anabaena PCC 7119 is used as a model system to investigate the fast internal dynamics of a flavin-bearing protein. Virtually complete backbone and side chain resonance NMR assignments of an oxidized flavodoxin point mutant (C55A) have been determined. Backbone and side chain dynamics in flavodoxin (C55A) were investigated using (15)N amide and deuterium methyl NMR relaxation methods. The squared generalized order parameters (S(NH)(2)) for backbone amide N-H bonds are found to be uniformly high ( approximately 0.923 over 109 residues in regular secondary structure), indicating considerable restriction of motion in the backbone of the protein. In contrast, methyl-bearing side chains are considerably heterogeneous in their amplitude of motion, as indicated by obtained symmetry axis squared generalized order parameters (S(axis)(2)). However, in comparison to nonprosthetic group-bearing proteins studied with these NMR relaxation methods, the side chains of oxidized flavodoxin are unusually rigid.     

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.     

Direct observation of protein-ligand interaction kinetics

Internal dynamics on the micro- to millisecond time scale have a strong influence on the affinity and specificity with which a protein binds ligands. This time scale is accessible through relaxation dispersion measurements using NMR. By studying the dynamics of a protein with different concentrations of a ligand, one can determine the dynamic effects induced by the ligand. Here we have studied slow internal dynamics of the N-terminal src homology 2 domain of phosphatidylinositide 3-kinase to probe the role of individual residues for the interaction with a tyrosine-phosphorylated binding sequence from polyoma middle T antigen. While slow dynamic motion was restricted to a few residues in the free SH2 and in the SH2 complex, motion was significantly enhanced by adding even small amounts of ligand. Kinetic rates induced by ligand binding varied between 300 and 2000 s(-1). High rates reflected direct interactions with the ligand or rearrangements caused by ligand binding. Large differences in rates were observed for residues adjacent in the primary sequence reflecting their individual roles in ligand interaction. However, rates were similar for residues involved in the same side chain interactions, reflecting concerted motions during ligand binding. For a subset of residues, exchange must involve structural intermediates which play a crucial role in high-affinity ligand binding. This analysis supports a new view of the dynamics of individual sites of a protein during ligand interaction.     

13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy

Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.     

Flexibility and ligand exchange in a buried cavity mutant of T4 lysozyme studied by multinuclear NMR

The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the wild-type protein by measuring the NMR spin relaxation rates of backbone amide and side chain methyl nuclei. Many residues surrounding the cavity were found to be affected by a chemical exchange process with a rate of 1500 +/- 200 s(-1), which is quenched upon addition of saturating amounts of the ligand xenon. The relationship between the structure, dynamics, and energetics of the T4 lysozyme mutant is discussed.     

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).     

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.     

Characterization of the backbone and side chain dynamics of the CaM-CaMKIp complex reveals microscopic contributions to protein conformational entropy

Calmodulin is a central mediator of calcium-dependent signal transduction pathways and regulates the activity of a large number of diverse targets. Calcium-dependent interactions of calmodulin with regulated proteins are of generally high affinity but of quite variable thermodynamic origins. Here we investigate the influence of the binding of the calmodulin-binding domain of calmodulin kinase I on the fast internal dynamics of calcium-saturated calmodulin. NMR relaxation was used to probe motion on the backbone (viewed through the backbone amide NH group) and the side chains (viewed through methyl groups). The distribution of the amplitudes of side chain dynamics is trimodal. The microscopic details of side chain motion are compared with those of a thermodynamically and structurally similar complex of calmodulin with the calmodulin-binding domain of the smooth muscle myosin light chain kinase. While there are no significant differences in backbone dynamics and no net change in methyl-bearing side chain dynamics, a large redistribution of the amplitude of methyl dynamics is observed between the two complexes. The variation in dynamics was largely localized to the heterogeneously dynamic target-binding interface, suggesting that differential dynamics of the binding surface plays a functional role in the high-affinity binding interactions of calmodulin. These results begin to reveal a fundamental role for residual protein entropy in molecular recognition by calmodulin.     

Measurement of 15N relaxation in deuterated amide groups in proteins using direct nitrogen detection

15N chemical shielding tensors contain useful structural information, and their knowledge is essential for accurate analysis of protein backbone dynamics. The anisotropic component (CSA) of 15N chemical shielding can be obtained from 15N relaxation measurements in solution. However, the predominant contribution to nitrogen relaxation from 15N-(1)H dipolar coupling in amide groups limits the sensitivity of these measurements to the actual CSA values. Here we present nitrogen-detected NMR experiments for measuring 15N relaxation in deuterated amide groups in proteins, where the dipolar contribution to 15N relaxation is significantly reduced by the deuteration. Under these conditions nitrogen spin relaxation becomes a sensitive probe for variations in 15N chemical shielding tensors. Using the nitrogen direct-detection experiments we measured the rates of longitudinal and transverse 15N relaxation for backbone amides in protein G in D(2)O at 11.7 T. The measured relaxation rates are validated by comparing the overall rotational diffusion tensor obtained from these data with that from the conventional 15N relaxation measurements in H(2)O. This analysis revealed a 17-24 degree angle between the NH-bond and the unique axis of the 15N chemical shielding tensor.     

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.     

Effect of pressure on the tertiary structure and dynamics of folded basic pancreatic trypsin inhibitor

The on-line high-pressure cell NMR technique was used to study pressure-induced changes in the tertiary structure and dynamics of a globular protein, basic pancreatic trypsin inhibitor (BPTI). Practically all the proton signals of BPTI were observed with (1)H two-dimensional NMR spectroscopy at 750 MHz at variable pressure between 1 and 2000 bar. Chemical shifts, nuclear Overhauser effect (NOE), and line shapes were used to analyze conformational and dynamic changes of the protein as functions of pressure. Linear, reversible, but nonuniform pressure-induced chemical shift changes of practically all the C(alpha) protons and side chain protons showed that the entire secondary and tertiary structures are altered by pressure within the folded ensemble of BPTI. The high field shift tendency of most side chain proton signals and the increase in NOE intensities of some specific side chain protons indicated a site-specific compaction of the tertiary structure. Pressure dependence of ring flip rates was deduced from resonance line shapes of the slices of the two-dimensional NMR spectrum for ring proton signals of Tyr-35 and Phe-45. The rates of the flip-flop motions were considerably reduced at high pressure, from which activation volumes were determined to be 85 +/- 20 A(3) (or 51.2 ml/mol) and 46 +/- 9 A(3) (or 27.7 ml/mol) for Tyr-35 and Phe-45, respectively, at 57 degrees C. The present experiments confirm that pressure affects the entire secondary and tertiary structures of a globular protein with specific compaction of a core, leading to quite significant changes in slow internal dynamics of a globular protein.     

An increase in side chain entropy facilitates effector binding: NMR characterization of the side chain methyl group dynamics in Cdc42Hs

Cdc42Hs is a signal transduction protein that is involved in cytoskeletal growth and organization. We describe here the methyl side chain dynamics of three forms of (2)H,(13)C,(15)N-Cdc42Hs [GDP-bound (inactive), GMPPCP-bound (active), and GMPPCP/PBD46-bound (effector-bound)] from (13)C-(1)H NMR measurements of deuterium T(1) and T(1 rho) relaxation times. A wide variation in flexibility was observed throughout the protein, with methyl axis order parameters (S(2)(axis)) ranging from 0.2 to 0.4 (highly disordered) in regions near the PBD46 binding site to 0.8--1.0 (highly ordered) in some helices. The side chain dynamics of the GDP and GMPPCP forms are similar, with methyl groups on the PBD46 binding surface experiencing significantly greater mobility (lower S(2)(axis)) than those not on the binding surface. Binding of PBD46 results in a significant increase in the disorder and a corresponding increase in entropy for the majority of methyl groups. Many of the methyl groups that experience an increase in mobility are found in residues that are not part of the PBD46 binding interface. This entropy gain represents a favorable contribution to the overall entropy of effector binding and partially offsets unfavorable entropy losses such as those that occur in the backbone.     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

NMR observable-based structure refinement of DAP12-NKG2C activating immunoreceptor complex in explicit membranes

NMR observables, such as NOE-based distance measurements, are increasingly being used to characterize membrane protein structures. However, challenges in membrane protein NMR studies often yield a relatively small number of such restraints that can create ambiguities in defining critical side chain-side chain interactions. In the recent solution NMR structure of the DAP12-NKG2C immunoreceptor transmembrane helix complex, five functionally required interfacial residues (two Asps and two Thrs in the DAP12 dimer and one Lys in NKG2C) display a surprising arrangement in which one Asp side chain faces the membrane hydrophobic core. To explore whether these side-chain interactions are energetically optimal, we used the published distance restraints for molecular dynamics simulations in explicit micelles and bilayers. The structures refined by this protocol are globally similar to the published structure, but the side chains of those five residues form a stable network of salt bridges and hydrogen bonds, leaving the Asp side chain shielded from the hydrophobic core, which is also consistent with available experimental observations. Moreover, the simulations show similar short-range interactions between the transmembrane complex and lipid/detergent molecules in micelles and bilayers, respectively. This study illustrates the efficacy of NMR membrane protein structure refinements in explicit membrane systems.     

Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex

The response of the internal dynamics of calcium-saturated calmodulin to the formation of a complex with a peptide model of the calmodulin-binding domain of the smooth muscle myosin light chain kinase has been studied using NMR relaxation methods. The backbone of calmodulin is found to be unaffected by the binding of the domain, whereas the dynamics of side chains are significantly perturbed. The changes in dynamics are interpreted in terms of a heterogeneous partitioning between structure (enthalpy) and dynamics (entropy). These data provide a microscopic view of the residual entropy of a protein in two functional states and suggest extensive enthalpy/entropy exchange during the formation of a protein-protein interface.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.