Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution.
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ABSTRACT:
SUBMITTER: Igumenova TI
PROVIDER: S-EPMC2547146 | biostudies-literature | 2006 May
REPOSITORIES: biostudies-literature
Publications
Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution.
Igumenova Tatyana I TI Frederick Kendra King KK Wand A Joshua AJ
Chemical reviews 20060501 5
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Project description: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.
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Project description:Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.
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Project description:Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin β TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin β(3)(Lys 716) helps determine β(3) TMD topography. The α(ΙΙb)β(3) TMD structure indicates that precise β(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the αβ TMD together to limit transmembrane signalling. Mutation of β(3)(Lys 716) caused dissociation of α(ΙΙb)β(3) TMDs and integrin activation. To confirm that altered topography of β(3)(Lys 716) mutants activated α(ΙΙb)β(3), we used directed evolution of β(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of β(3) TMD (A711P) increased α(ΙΙb)β(3) TMD association and inactivated integrin α(ΙΙb)β(3)(A711P,K716A). β(3)(Pro 711) introduced a TMD kink of 30 ± 1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.
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Project description:Histidine is a key amino-acid residue in proteins with unique properties engendered by its imidazole side chain that can exist in three different states: two different neutral tautomeric forms and a protonated, positively charged one with a pKa value close to physiological pH. Commonly, two or all three states coexist and interchange rapidly, enabling histidine to act as both donor and acceptor of hydrogen bonds, coordinate metal ions, and engage in acid/base catalysis. Understanding the exchange dynamics among the three states is critical for assessing histidine's mechanistic role in catalysis, where the rate of proton exchange and interconversion among tautomers might be rate limiting for turnover. Here, we determine the exchange kinetics of histidine residues with pKa values representative of the accessible range from 5 to 9 by measuring pH-dependent 15N, 13C, and 1H transverse relaxation rate constants for 5 nuclei in each imidazole. Proton exchange between the imidazole and the solvent is mediated by hydronium ions at acidic and neutral pH, whereas hydroxide mediated exchange becomes the dominant mechanism at basic pH. Proton transfer is very fast and reaches the diffusion limit for pKa values near neutral pH. We identify a direct pathway between the two tautomeric forms, likely mediated by a bridging water molecule or, in the case of high pH, hydroxide ion. For histidines with pKa 7, we determine all rate constants (lifetimes) involving protonation over the entire pH range. Our approach should enable critical insights into enzymatic acid/base catalyzed reactions involving histidines in proteins.
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Project description:Coordination of proteins and peptides to metal ions is known to affect their properties, often by a change in their structural organization. Side chains of the residues directly involved in metal binding or very close to the coordination centre may arrange themselves around it, in such a way that they can, for instance, disrupt the protein functions or stabilize a metal complex by shielding it from the attack of water or other small molecules. The conformation of these side chains may be crucial to different biological or toxic processes. In our research we have encountered such behaviour in several cases, leading to interesting results for our purposes. Here we give an overview on the structural changes involving peptide side chains induced by Ni(II) coordination. In this paper we deal with a number of peptides, deriving from proteins containing one or more metal coordinating sites, which have been studied through a series of NMR experiments in their structural changes caused by Ni(II) complexation. Several peptides have been included in the study: short sequences from serum albumin (HSA), Des-Angiotensinogen, the 30-amino acid tail of histone H4, some fragments from histone H2A and H2B, the initial fragment of human protamine HP2 and selected fragments from prion and Cap43 proteins. NMR was the election technique for gathering structural information. Experiments performed for this purpose included 1D ¹H and ¹³C, and 2D HSQC, COSY, TOCSY, NOESY and ROESY acquisitions, which allowed the calculation of the Ni(II) complexes structural models.
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Project description:Many ions are known to affect the activity, stability, and structural integrity of proteins. Although this effect can be generally attributed to ion-induced changes in forces that govern protein folding, delineating the underlying mechanism of action still remains challenging because it requires assessment of all relevant interactions, such as ion-protein, ion-water, and ion-ion interactions. Herein, we use two unnatural aromatic amino acids and several spectroscopic techniques to examine whether guanidinium chloride, one of the most commonly used protein denaturants, and tetrapropylammonium chloride can specifically interact with aromatic side chains. Our results show that tetrapropylammonium, but not guanidinium, can preferentially accumulate around aromatic residues and that tetrapropylammonium undergoes a transition at ∼1.3 M to form aggregates. We find that similar to ionic micelles, on one hand, such aggregates can disrupt native hydrophobic interactions, and on the other hand, they can promote α-helix formation in certain peptides.
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Project description:Potentials of mean force (PMF) between ionizable amino acid side chains (Arg, Lys, His, Glu) in the headgroup area of a palmitoyl oleoyl phosphatidylcholine lipid bilayer were obtained from all-atom molecular dynamics simulations and the adaptive biasing force method. Simulations in bulk water were also performed for comparison. Side chains were constrained in collinear, stacking, and orthogonal (T-shaped) orientations. The most structured and attractive PMFs were observed for hydrogen-bonded side chains. Contact minima occurred at a distance of 2.6-3.1 Å between selected atoms or centers of mass with the most attractive interaction (-9.6 kcal/mol) observed between Arg(+) and Glu(-). Hydrogen bonds play a significant role in stabilizing these interactions. Interactions between like charged side chains can also be very attractive if the charges are screened by surrounding molecules or groups (e.g., the PMF value at the contact minimum for Arg(+)···Arg(+) is -7.6 kcal/mol). Like charged side chains can have contact minima as close as 3.6 Å. The PMFs depend strongly on the relative orientation of the side chains. In agreement with experimental studies and other simulations, we found the stacking arrangement of like charged side chains to be the most favorable orientation. Interaction energies and Lennard-Jones energies between side chains, headgroups, and water molecules were analyzed in order to rationalize the observed PMFs and their dependence on orientation. In general, the results cannot be explained by simple dielectric arguments.
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Project description:The influence of lone-pair electrons on the directionality of hydrogen bonds that are formed by oxygen and nitrogen atoms in the side chains of nine hydrophilic was investigated using molecular dynamics simulations. The simulations were conducted using two types of force fields; one incorporated lone-pair electrons placed at off-atom sites and the other did not. The density distributions of the hydration water molecules around the oxygen and nitrogen atoms were calculated from the simulation trajectories, and were compared with the empirical hydration distribution functions, which were constructed from a large number of hydration water molecules found in the crystal structures of proteins. Only simulations using the force field explicitly incorporating lone-pair electrons reproduced the directionality of hydrogen bonds that is observed in the empirical distribution functions for the deprotonated oxygen and nitrogen atoms in the sp 2-hybridization. The amino acids that include such atoms are functionally important glutamate, aspartate, and histidine. Therefore, a set of force field that incorporates lone-pair electrons as off-atom charge sites would be effective for considering hydrogen bond formation by these amino acids in molecular dynamics simulation studies.
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Project description:The conformational dynamics of a pathogenic κ4 human immunoglobulin light-chain variable domain, SMA, associated with AL amyloidosis, were investigated by 15N relaxation dispersion NMR spectroscopy. Compared to a homologous light-chain, LEN, which differs from SMA at eight positions but is non-amyloidogenic in vivo, we find that multiple residues in SMA clustered around the N-terminus and CDR loops experience considerable conformational exchange broadening caused by millisecond timescale protein motions, consistent with a destabilized dimer interface. To evaluate the contribution of each amino acid substitution to shaping the dynamic conformational landscape of SMA, NMR studies were performed for each SMA-like point mutant of LEN followed by in silico analysis for a subset of these proteins. These studies show that a combination of only three mutations located within or directly adjacent to CDR3 loop at the dimer interface, which remarkably include both destabilizing (Q89H and Y96Q) and stabilizing (T94H) mutations, largely accounts for the differences in conformational flexibility between LEN and SMA. Collectively, our studies indicate that a correct combination of stabilizing and destabilizing mutations is key for immunoglobulin light-chains populating unfolded intermediates that result in amyloid formation, and underscore the complex nature of correlations between light-chain conformational flexibility, thermodynamic stability and amyloidogenicity.
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