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PubMed2006

Accessibility of cysteine residues substituted into the cytoplasmic regions of the alpha-factor receptor identifies the intracellular residues that are available for G protein interaction.

The yeast alpha-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 was substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEA-biotin [[2-[(biotinoyl)amino]ethyl]methanethiosulfonate], which reacts with solvent-accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH >or=7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.

Amino Acid Sequence↗

PubMed2006

Analysis of sugar bioprotective mechanisms on the thermal denaturation of lysozyme from Raman scattering and differential scanning calorimetry investigations.

Sugar-induced thermostabilization of lysozyme was analyzed by Raman scattering and modulated differential scanning calorimetry investigations, for three disaccharides (maltose, sucrose, and trehalose) characterized by the same chemical formula (C(12)H(22)O(11)). This study shows that trehalose is the most effective in stabilizing the folded secondary structure of the protein. The influence of sugars on the mechanism of thermal denaturation was carefully investigated by Raman scattering experiments carried out both in the low-frequency range and in the amide I band region. It was determined that the thermal stability of the hydrogen-bond network of water, highly dependent on the presence of sugars, contributes to the stabilization of the native tertiary structure and inhibits the first stage of denaturation, that is, the transformation of the tertiary structure into a highly flexible state with intact secondary structure. It was found that trehalose exhibits exceptional capabilities to distort the tetra-bonded hydrogen-bond network of water and to strengthen intermolecular O-H interactions responsible for the stability of the tertiary structure. Trehalose was also observed to be the best stabilizer of the folded secondary structure, in the transient tertiary structure, leading to a high-temperature shift of the unfolding process (the second stage of denaturation). This was interpreted from the consideration that the transient tertiary structure is less flexible and inhibits the solvent accessibility around the hydrophobic groups of lysozyme.

Amides↗

PubMed1992

Localization of bound water in the solution structure of the immunoglobulin binding domain of streptococcal protein G. Evidence for solvent-induced helical distortion in solution.

The presence of bound water in the solution structure of the IgG binding domain of streptococcal protein G has been investigated by nuclear magnetic resonance using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. The backbone amide protons of three residues, Ala20, Gln32 and Tyr33, are found to be in close proximity to bound water. Examination of the three-dimensional structure of the IgG binding domain indicates that in the vicinity of these three residues there are no backbone groups that do not already participate in hydrogen bonding and there are no suitably placed side-chain groups available for hydrogen bonding with water. As the lifetime of the bound water detected in this nuclear magnetic resonance experiment is greater than about one nanosecond, it is likely that the two bound water molecules participate in a bifurcating hydrogen bonding network comprising a CO-NH hydrogen bonded pair, such that the water molecule accepts a hydrogen bond from the NH proton and donates one to the carbonyl oxygen with the result that the amide proton is involved in a three center hydrogen bond. On the basis of the structure, one water molecule participates in such an interaction with the Ala20(NH)-Met1(CO) hydrogen bonded pair at the beginning of an anti-parallel beta-sheet, and the other with the Tyr33(NH)-Val29(CO) hydrogen bonded pair in the single alpha-helix. The latter, which is external and solvent accessible, is associated with a distortion in the alpha-helix centered around Tyr33 which consists of a significant increase in the CO(i-4)-N(i) and CO(i-4)-NH(i) distances relative to those in the rest of the helix, as well as a significant departure in the phi, psi angles of Tyr33 relative to regular helical geometry. Such solvent induced distortions in alpha-helices have been previously noticed in crystal structures and were postulated as possible folding intermediates for helical structures. The present observation of this phenomenon in solution indicates, however, that these water molecules are tightly bound and represent an integral part of the protein framework.

Bacterial Proteins↗

PubMed2004

Transmembrane domain 5 of the LdNT1.1 nucleoside transporter is an amphipathic helix that forms part of the nucleoside translocation pathway.

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donovani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M(176)C, T(186)C, S(187)C, Q(190)C, V(193)C, and K(194)C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q(190)C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic alpha-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.

Adenosine↗

PubMed2004

Anchor residues in protein-protein interactions.

We show that the mechanism for molecular recognition requires one of the interacting proteins, usually the smaller of the two, to anchor a specific side chain in a structurally constrained binding groove of the other protein, providing a steric constraint that helps to stabilize a native-like bound intermediate. We identify the anchor residues in 39 protein-protein complexes and verify that, even in the absence of their interacting partners, the anchor side chains are found in conformations similar to those observed in the bound complex. These ready-made recognition motifs correspond to surface side chains that bury the largest solvent-accessible surface area after forming the complex (> or =100 A2). The existence of such anchors implies that binding pathways can avoid kinetically costly structural rearrangements at the core of the binding interface, allowing for a relatively smooth recognition process. Once anchors are docked, an induced fit process further contributes to forming the final high-affinity complex. This later stage involves flexible (solvent-exposed) side chains that latch to the encounter complex in the periphery of the binding pocket. Our results suggest that the evolutionary conservation of anchor side chains applies to the actual structure that these residues assume before the encounter complex and not just to their loci. Implications for protein docking are also discussed.

Antigen-Antibody Complex↗

PubMed1996

Solid state and solution conformations of a helical peptide with a central Gly-Gly segment.

The influence of amino acids with contrasting conformational tendencies on the stereochemistry of oligopeptides has been investigated using an octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe, which contains two helix-promoting Aib residues and a central helix-destabilizing Gly-Gly segment. Single crystal x-ray diffraction studies reveal that a 3(10)-helix is formed up to the penultimate Aib residue, at which point there is a helix reversal in the backbone, reminiscent of a C-terminal 6-->1 hydrogen bond. The curious feature in the crystal is the solvation of the possible 6-->1 bond by a CH3OH molecule, where the OH is inserted between O(3) and N(8) and participates in hydrogen bonds with both. The cell parameters are as follows: space group P2(1)2(1)2(1), a = 10.649 (4) A, b = 15.694 (5) A, c = 30.181 (8) A, R = 6.7% for 3427 data (magnitude of F0 > 3 sigma F) observed to 0.9 A. Nuclear magnetic resonance studies in CDCl3 using NH group solvent accessibility and nuclear Overhauser effects as probes are consistent with a 3(10)-helical conformation. In contrast, in (CD3)2SO, unfolding of the central segment results in a multiple beta-turn structure, with beta-turn conformations populated at residues 1-2, 3-4, and 6-7. CD studies in methanol-2,2,2-trifluoroethanol (TFE) mixtures also provide evidence for a solvent-dependent structural transition. Helical conformations are populated in TFE, while type II beta-turn structures are favored in methanol.

Chemical Phenomena↗

PubMed1999

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins.

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.

Abrin↗

PubMed2001

A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.

Bacillus cereus↗

PubMed2005

Probing protein tertiary structure with amidination.

A chemical derivatization method, amidination, that has recently been effectively employed in peptide mass spectrometry experiments is used to covalently modify lysines in several standard proteins. Protein and peptide mass spectra identify sites at which the reaction does or does not occur. This is therefore a rapid approach to elucidate solvent-accessible regions of folded proteins.

Amination↗

PubMed1996

A ligand-gated, hinged loop rearrangement opens a channel to a buried artificial protein cavity.

Conformational changes that gate the access of substrates or ligands to an active site are important features of enzyme function. In this report, we describe an unusual example of a structural rearrangement near a buried artificial cavity in cytochrome c peroxidase that occurs on binding protonated benzimidazole. A hinged main-chain rotation at two residues (Pro 190 and Asn 195) results in a surface loop rearrangement that opens a large solvent-accessible channel for the entry of ligands to an otherwise inaccessible binding site. The trapping of this alternate conformational state provides a unique view of the extent to which protein dynamics can allow small molecule penetration into buried protein cavities.

Crystallography, X-Ray↗

PubMed2002

Solvation model dependency of helix-coil transition in polyalanine.

Helix-coil transitions in polyalanine molecules of length 10 are studied by multi-canonical Monte Carlo simulations. The solvation effects are included by either a distance-dependent dielectric permittivity or by a term that is proportional to the solvent-accessible surface area of the peptide. We found a strong dependence of the characteristics of the helix-coil transition from the details of the solvation model.

Biophysical Phenomena↗

PubMed2006

Specific radiation damage to acidic residues and its relation to their chemical and structural environment.

Intense synchrotron radiation produces specific structural and chemical damage to crystalline proteins even at 100 K. Carboxyl groups of acidic residues (Glu, Asp) losing their definition is one of the major effects observed. Here, the susceptibilities to X-ray damage of acidic residues in tetrameric malate dehydrogenase from Haloarcula marismortui are investigated. The marked excess of acidic residues in this halophilic enzyme makes it an ideal target to determine how specific damage to acidic residues is related to their structural and chemical environment. Four conclusions are drawn. (i) Acidic residues interacting with the side-chains of lysine and arginine residues are less affected by radiation damage than those interacting with serine, threonine and tyrosine side-chains. This suggests that residues with higher pK(a) values are more vulnerable to damage than those with a lower pK(a). However, such a correlation was not found when calculated pK(a) values were inspected. (ii) Acidic side-chains located in the enzymatic active site are the most radiation-sensitive ones. (iii) Acidic residues in the internal cavity formed by the four monomers and those involved in crystal contacts appear to be particularly susceptible. (iv) No correlation was found between radiation susceptibility and solvent accessibility.

Amino Acids↗

PubMed1998

Kinetics of lysozyme refolding: structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering.

We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.

Animals↗

PubMed2005

Effect of pressure on the conformation of proteins. A molecular dynamics simulation of lysozyme.

The effect of pressure on the structure and mobility of lysozyme was studied by molecular dynamics computer simulation at 1 and 3 kbar (1 atm = 1.01325 bar = 101.325 kPa). The results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. From the analysis of secondary structure along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. The difference in solvent-accessed surface (SAS) with pressure shows a clear inversion of the hydrophilic/hydrophobic SAS ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

Carbon↗

PubMed1999

A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease.

Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.

Amino Acid Substitution↗

PubMed2006

Analysis of the oxidative damage-induced conformational changes of apo- and holocalmodulin by dose-dependent protein oxidative surface mapping.

Calmodulin (CaM) is known to undergo conformational and functional changes on oxidation, allowing CaM to function as an oxidative stress sensor. We report the use of a novel mass spectrometry-based methodology to monitor the structure of apo- and holo-CaM as it undergoes conformational changes as a result of increasing amounts of oxidative damage. The kinetics of oxidation for eight peptides are followed by mass spectrometry, and 12 sites of oxidation are determined by MS/MS. Changes in the pseudo-first-order rate constant of oxidation for a peptide after increasing radiation exposure reveal changes in the accessibility of the peptide to the diffusing hydroxyl radical, indicating conformational changes as a function of increased oxidative damage. For holo-CaM, most sites rapidly become less exposed to hydroxyl radicals as the protein accumulates oxidative damage, indicating a closing of the hydrophobic pockets in the N- and C-terminal lobes. For apo-CaM, many of the sites rapidly become more exposed until they resemble the solvent accessibility of holo-CaM in the native structure and then rapidly become more buried, mimicking the conformational changes of holo-CaM. At the most heavily damaged points measured, the rates of oxidation for both apo- and holo-CaM are essentially identical, suggesting the two assume similar structures.

Amino Acid Sequence↗

PubMed2002

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy.

Ubiquitination is a post-translation modification whereby the C-terminal end of ubiquitin (Ub) is covalently attached to the amino group of a lysine in a target protein. Additional ubiquitin groups are added using Ub-Ub linkages to form a polyubiquitin chain. A 26S protease complex specifically binds polyubiquitinated proteins and degrades them in an ATP-dependent manner. The target lysine in the substrate protein resides in a domain that is recognized by the ubiquitination machinery in a temporally and spatially controlled manner. The accessibility and the molecular dynamics of the target domain for each protein substrate is expected to be distinctive and this article is intended to facilitate investigations in this uncharted research area of ubiquitination mediated protein turnover by means of site-directed spin labeling. Examples illustrate the methodology of electron paramagnetic resonance data acquisition and interpretation in terms of secondary and tertiary structure resolution of proteins and protein complexes. Analysis of the spin labeled side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin labels allow to model protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. The structural changes accompanying protein function or protein-protein interaction can be monitored in the millisecond time range. These features make site-directed spin labeling an attractive approach for the study of protein--ubiquitin interaction and protein ubiquitination.

Computer Simulation↗

PubMed1996

Prediction and evaluation of side-chain conformations for protein backbone structures.

A common approach to protein modeling is to propose a backbone structure based on homology or threading and then to attempt to build side chains onto this backbone. A fast algorithm using the simple criteria of atomic overlap and overall rotamer probability is proposed for this purpose. The method was first tested in the context of exhaustive searches of side chain configuration space in protein cores and was then applied to all side chains in 49 proteins of known structure, using simulated annealing to sample space. The latter procedure obtains the correct rotamer for 57% and the correct chi 1 value for 74% of the 6751 residues in the sample. When low-temperature Monte-Carlo simulations are initiated from the results of the simulated-annealing processes, consensus configurations are obtained which exhibit slightly more accurate predictions. The Monte-Carlo procedure also allows converged side chain entropies to be calculated for all residues. These prove to be accurate indicators of prediction reliability. For example, the correct rotamer is obtained for 79% and the correct chi 1 value is obtained for 84% of the half of the sample residues exhibiting the lowest entropies. Side chain entropy and predictability are nearly completely uncorrelated with solvent-accessible area. Some precedents for and implications of this observation are discussed.

Algorithms↗