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Results for “conformational ensemble”

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PubMed2003

Structure and polymorphism of the principal neutralization site of Thailand HIV-1 isolate.

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significantly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resolution 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the "global" structure which gives rise to inverse gamma-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.

Cystine↗

PubMed2005

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.

Animals↗

PubMed1999

The solution structure of a superpotent B-chain-shortened single-replacement insulin analogue.

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.

Circular Dichroism↗

PubMed1999

The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli.

The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincón G et al., 1993, Biochemistry 32:13,981-13,990). To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS.

Circular Dichroism↗

PubMed1982

Statistical molecular model for hydrated peptides. Application to angiotensin II analogs.

A statistical molecular model for hydrated peptides is used to stimulate the behaviour of various peptide chains in aqueous solution. The reliability of the model is ascertained by the evolution of the characteristic ratio, Cn, for hydrophobic and hydrophilic polypeptide chains, such as, respectively, poly-Ala and poly-Ser. The comparison of computed and experimental properties related to non radiative energy transfer of angiotensin II analogs, assuming that an ensemble of conformers is a satisfactory representation of the state of these molecules in water, provides further support for the model.

Amino Acid Sequence↗

PubMed2005

Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach.

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 A greater than that of the native state, contain on average 37%beta-sheet secondary structure. In these conformers the central regions of the two orthogonal beta-sheets that make up the beta-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the beta-sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85-104 (beta-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface.

Computer Simulation↗

PubMed2006

Optimization and computational evaluation of a series of potential active site inhibitors of the V82F/I84V drug-resistant mutant of HIV-1 protease: an application of the relaxed complex method of structure-based drug design.

The Relaxed Complex method, an approach to structure-based drug design that incorporates the flexibilities of both the ligand and target protein, was applied to the immunodeficiency virus protease system. The control cases used AutoDock3.0.5 to dock a fully flexible version of the prospective drug JE-2147 (aka SM-319777 or KNI-764) to large ensembles of conformations extracted from conventional, all atom, explicitly solvated molecular dynamic simulations of the wild type, and the V82F/I84V drug-resistant mutant of HIV-1 protease. The best set of run parameters from the control cases produced robust results when used against 2200 different conformations of the wild-type HIV-1 protease or against 2200 conformations of the mutant. The results of the control cases, the published advice from experts, and structural intuition were used to design a new series of 23 potential active site inhibitors. The compounds were evaluated by docking them against 700 different conformations of the V82F/I84V mutant. The results of this first round of lead optimization were quite promising. Approximately one-third of that series performed at least slightly better than the parent compound, and four of those compounds displayed significantly better binding affinities against that drug-resistant mutant (within our computational model).

Amino Acid Substitution↗

PubMed2005

Exploring the helix-coil transition via all-atom equilibrium ensemble simulations.

The ensemble folding of two 21-residue alpha-helical peptides has been studied using all-atom simulations under several variants of the AMBER potential in explicit solvent using a global distributed computing network. Our extensive sampling, orders of magnitude greater than the experimental folding time, results in complete convergence to ensemble equilibrium. This allows for a quantitative assessment of these potentials, including a new variant of the AMBER-99 force field, denoted AMBER-99 phi, which shows improved agreement with experimental kinetic and thermodynamic measurements. From bulk analysis of the simulated AMBER-99 phi equilibrium, we find that the folding landscape is pseudo-two-state, with complexity arising from the broad, shallow character of the "native" and "unfolded" regions of the phase space. Each of these macrostates allows for configurational diffusion among a diverse ensemble of conformational microstates with greatly varying helical content and molecular size. Indeed, the observed structural dynamics are better represented as a conformational diffusion than as a simple exponential process, and equilibrium transition rates spanning several orders of magnitude are reported. After multiple nucleation steps, on average, helix formation proceeds via a kinetic "alignment" phase in which two or more short, low-entropy helical segments form a more ideal, single-helix structure.

Algorithms↗

PubMed2000

Multi-dimensional QSAR in drug research. Predicting binding affinities, toxicity and pharmacokinetic parameters.

Quantitative structure-activity relationships (QSAR) are an area of computational research which builds atomistic or virtual models to predict the biological activity or the toxicity of known or hypothetical substances. Of particular interest for the biomedical sciences are three-dimensional receptor surrogates (3D-QSAR) because they allow for the simulation of directional forces such as hydrogen bonds or metal-ligand contacts--key interactions for both molecular recognition and stereospecific ligand binding. While more powerful approaches make use of a genetic algorithm or a neural network to evolve a receptor surrogate, its predictive power still critically depends on the spatial alignment of the ligand molecules--mirroring the pharmacophore hypothesis--used to construct it. To avoid this bias, a recent development at our laboratory includes the possibility to represent each ligand molecule by an ensemble of conformations, orientations and protonation states as the fourth dimension (4D-QSAR). In addition, it allows for a potentially flexible receptor site (mimicking local induced fit) and solvent-accessible or shallow binding pockets. In this account, we seek to document the superiority of 4D-QSAR compared to 3D-concepts with simulations on the steroid, the aryl hydrocarbon and the neurokinin-1 receptor system. More complex, future applications of 4D-QSAR--the establishment of a virtual laboratory for the assessment of receptor-mediated toxicity and the prediction of oral bioavailability--are outlined.

Animals↗

PubMed2004

Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble.

A multi-site, time-resolved fluorescence resonance energy transfer methodology has been used to study structural heterogeneity in a late folding intermediate ensemble, IL, of the small protein barstar. Four different intra-molecular distances have been measured within the structural components of IL. The IL ensemble is shown to consist of different sub-populations of molecules, in each of which one or more of the four distances are native-like and the remaining distances are unfolded-like. In very stable conditions that favor formation of IL, all four distances are native-like in most molecules. In less stable conditions, one or more distances are unfolded-like. As stability is decreased, the proportion of molecules with unfolded-like distances increases. Thus, the results show that protein folding intermediates are ensembles of different structural forms, and they demonstrate that conformational entropy increases as structures become less stable. These observations provide direct experimental evidence in support of a basic tenet of energy landscape theory for protein folding, that available conformational space, as represented by structural heterogeneity in IL, becomes restricted as the stability is increased. The results also vindicate an important prediction of energy landscape theory, that different folding pathways may become dominant under different folding conditions. In more stable folding conditions, uniformly native-like compactness is achieved during folding to IL, whereas in less stable conditions, uniformly native-like compactness is achieved only later during the folding of IL to N.

Bacterial Proteins↗

PubMed1999

Side-chain conformational disorder in a molten globule: molecular dynamics simulations of the A-state of human alpha-lactalbumin.

Molten globules are compact, partially folded forms of proteins consisting of an ensemble of interconverting conformers with disorder in the side-chain packing across the ensemble. Using insights from experimental data a strategy has been devised to describe this side-chain disorder in a molten globule. Structures of human alpha-lactalbumin have been generated with significantly different side-chain packings to those observed in the native protein by changing all the chi1 torsion angles. Molecular dynamics simulations have been carried out starting from these structures using conditions under which the molten globule is seen experimentally, i.e. at low pH and in the absence of calcium. In each simulation, low energy conformers were generated which are compact and retain substantial secondary structure and an overall native fold, but have very different side-chain contacts; there are all-atom root-mean-square differences of up to 0.5 nm between the different structures. The structures from the different simulations taken together provide an initial description of possible contributors to the ensemble of conformers in the molten globule state which is consistent, at least in general terms, with experimental data.

Calcium↗

PubMed1993

NMR solution structure and flexibility of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa.

A synthetic peptide antigen corresponding to the C-terminus of Pseudomonas aeruginosa K strain pilin has been studied by one and two-dimensional NMR techniques. This peptide exists in two isomeric forms which arise as a result of the I138-P139 amide bond. An ensemble of solution conformations for the trans form of this 17-residue disulfide-bridged peptide (PAK 128-144) has been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. One major class of backbone conformations has been identified for this potential synthetic vaccine and indicates the presence of two beta-turns in the region 134-142. The region that has been established as the epitope for the monoclonal antibody PK99H is consistent with the region of the major conformers that exhibit the most definition in the ensemble (134-140) and also includes a type I beta-turn from residues 134 to 137. The generated structures are also consistent with observed NOEs characteristic of beta-turns and amide proton temperature coefficient data, which indicate the presence of two turns between residues 134 and 142. The presence of secondary structure within the epitope substantiates the theory that immunogenic regions of proteins are those which contain surface-exposed structural elements such as beta-turns. Further implications of the structure on antigenicity and cross-reactivity are discussed.

Amino Acid Sequence↗

PubMed2004

Structural basis for the attachment of a paramyxoviral polymerase to its template.

The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain. We have investigated the interaction between the binding domain from measles P (amino acids 457-507) and the sequence it recognizes within measles N (amino acids 477-505). By using both solution NMR spectroscopy and x-ray crystallography, we show that N(487-503) binds as a helix to the surface created by the second (alpha2) and third (alpha3) helices of P(457-507), in an orientation parallel to the helix alpha3, creating a four-helix bundle. The binding interface is tightly packed and dominated by hydrophobic amino acids. Binding and folding of N(487-503) are coupled. However, when not bound to P, N(487-503) does not resemble a statistical random coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N(487-503) is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase-template interactions in a paramyxovirus and presents an example of a protein-protein interaction that must be only transiently maintained as part of its normal function.

Binding Sites↗

PubMed2005

Transient interaction with nanoparticles "freezes" a protein in an ensemble of metastable near-native conformations.

It is well-known that adsorption of proteins on interfaces often induces substantial alterations of the protein structure. However, very little is known about whether these conformational changes have any consequence for the protein conformation after desorption from the interface. To investigate this matter, we have selected a protein-particle system in which the enzyme human carbonic anhydrase I (HCAI) alternates between the adsorbed and free state upon interaction with the silica nanoparticles. High-resolution NMR analysis of the protein with the particles present in the sample shows a spectrum that indicates a molten globular-like structure. Removal of particles results in refolding of virtually all HCAI molecules to a fully active form. However, the two-dimensional NMR analysis shows that refolding does not result in a single well-defined protein structure but rather provides an ensemble of protein molecules with near-native conformations. A detailed comparative chemical shift analysis of 108 amide signals in (1)H-(15)N HSQC spectra of native and desorbed HCAI reveals that the most profound effects are located at beta-strands in the center of the molecule. The observation of very slow H-D exchange in the central beta-strands of HCAI [Kjellsson, A., Sethson, I., and Jonsson, B. H. (2003) Biochemistry 42, 363-374] in conjunction with our results indicates that the kinetic barriers for conformational rearrangements in the central core of the protein are low in the presence of nanoparticles but are very high under native conditions.

Carbonic Anhydrase I↗

PubMedDate not supplied

[Effect of intramolecular energetic barriers on conformation-non-equilibrium states of protein globules in ensemble].

Within the Blumenfeld's model interpretation theoretical investigations of conformation relaxation of the protein globule were carried out. Analytical expressions describing the relaxation time and cyclic frequency of enzymes synchronization by arbitrary number of metastable states and unequal probabilities of transitions between them were found. It was shown that inequality of activation energies affects the frequency and number of cycles of enzymes synchronous work to a considerably greater degree, than the relaxation time of initial disturbance. The number of macromolecules in the definite conformation state was evaluated and conditions of experimental observation of synchronization were discussed.

Biophysical Phenomena↗

PubMed1999

Exploring the dynamic information content of a protein NMR structure: comparison of a molecular dynamics simulation with the NMR and X-ray structures of Escherichia coli ribonuclease HI.

The multiconformer nature of solution nuclear magnetic resonance (NMR) structures of proteins results from the effects of intramolecular dynamics, spin diffusion and an uneven distribution of structural restraints throughout the molecule. A delineation of the former from the latter two contributions is attempted in this work for an ensemble of 15 NMR structures of the protein Escherichia coli ribonuclease HI (RNase HI). Exploration of the dynamic information content of the NMR ensemble is carried out through correlation with data from two crystal structures and a 1.7-ns molecular dynamics (MD) trajectory of RNase HI in explicit solvent. Assessment of the consistency of the crystal and mean MD structures with nuclear Overhauser effect (NOE) data showed that the NMR ensemble is overall more compatible with the high-resolution (1.48 A) crystal structure than with either the lower-resolution (2.05 A) crystal structure or the MD simulation. Furthermore, the NMR ensemble is found to span more conformational space than the MD simulation for both the backbone and the sidechains of RNase HI. Nonetheless, the backbone conformational variability of both the NMR ensemble and the simulation is especially consistent with NMR relaxation measurements of two loop regions that are putative sites of substrate recognition. Plausible side-chain dynamic information is extracted from the NMR ensemble on the basis of (i) rotamericity and syn-pentane character of variable torsion angles, (ii) comparison of the magnitude of atomic mean-square fluctuations (msf) with those deduced from crystallographic thermal factors, and (iii) comparison of torsion angle conformational behavior in the NMR ensemble and the simulation. Several heterogeneous torsion angles, while adopting non-rotameric/syn-pentane conformations in the NMR ensemble, exist in a unique conformation in the simulation and display low X-ray thermal factors. These torsions are identified as sites whose variability is likely to be an artifact of the NMR structure determination procedure. A number of other torsions show a close correspondence between the conformations sampled in the NMR and MD ensembles, as well as significant correlations among crystallographic thermal factors and atomic msf calculated from the NMR ensemble and the simulation. These results indicate that a significant amount of dynamic information is contained in the NMR ensemble. The relevance of the present findings for the biological function of RNase HI, protein recognition studies, and previous investigations of the motional content of protein NMR structures are discussed.

Cluster Analysis↗

PubMed1984

[Statistical weights of conformers of thyroliberin molecule and the averaging of its calculated structural characteristics].

A statistical ensemble of thyroliberin conformations has been generated using Monte-Carlo techniques. The averaged vicinal coupling constants for the peptide backbone and His side chain, percentage of prolyl cis-isomers and characteristics of possible intramolecular hydrogen bonds have been calculated. The obtained mean values are in good agreement with the experimental results. This fact indicates that the generated statistical ensemble is similar to the true one and evidences against the existence in solution of a single unique structure of thyroliberin.

Monte Carlo Method↗

PubMed1997

Factors affecting the ability of energy functions to discriminate correct from incorrect folds.

Eighteen low and medium resolution empirical energy functions were tested for their ability to distinguish correct from incorrect folds from three test sets of decoy protein conformations. The energy functions included 13 pairwise potentials of mean force, covering a wide range of functional forms and methods of parameterization, four potentials that attempt to detect properly formed hydrophobic cores, and one environment-based potential. the first of the three test sets consists of large ensembles of plausible conformations for eight small proteins, all of which have correct native secondary structure and are reasonably compact. The second is the set of all subconformations in a database of known protein structures applied to the sequences in that database (ungapped threading). The third is a set of ensembles of 1000 conformations each for seven small proteins taken from molecular dynamics simulations at 298 K and 498 K. Our results show that there are functions effective for each challenge set; moreover, success in one test is no guarantee of success in another. We examine the factors that seem to be important for accurate discrimination of correct structures in each of the test sets, and note that extremely simple functions are often as effective as more complex functions.

Chemical Phenomena↗