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Results for “conformational ensemble”

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PubMed2006

Simple estimation of absolute free energies for biomolecules.

One reason that free energy difference calculations are notoriously difficult in molecular systems is due to insufficient conformational overlap, or similarity, between the two states or systems of interest. The degree of overlap is irrelevant, however, if the absolute free energy of each state can be computed. We present a method for calculating the absolute free energy that employs a simple construction of an exactly computable reference system which possesses high overlap with the state of interest. The approach requires only a physical ensemble of conformations generated via simulation and an auxiliary calculation of approximately equal central-processing-unit cost. Moreover, the calculations can converge to the correct free energy value even when the physical ensemble is incomplete or improperly distributed. As a "proof of principle," we use the approach to correctly predict free energies for test systems where the absolute values can be calculated exactly and also to predict the conformational equilibrium for leucine dipeptide in implicit solvent.

Algorithms↗

PubMed2006

Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin.

The native states of proteins exist as an ensemble of conformationally similar microstates. The fluctuations among different microstates are of great importance for the functions and structural stability of proteins. Here, we demonstrate that single molecule atomic force microscopy (AFM) can be used to directly probe the existence of multiple folded microstates. We used the AFM to repeatedly stretch and relax a recombinant tenascin fragment TNfnALL to allow the fibronectin type III (FnIII) domains to undergo repeated unfolding/refolding cycles. In addition to the native state, we discovered that some FnIII domains can refold from the unfolded state into a previously unrecognized microstate, N* state. This novel state is conformationally similar to the native state, but mechanically less stable. The native state unfolds at approximately 120 pN, while the N* state unfolds at approximately 50 pN. These two distinct populations of microstates constitute the ensemble of the folded states for some FnIII domains. An unfolded FnIII domain can fold into either one of the two microstates via two distinct folding routes. These results reveal the dynamic and heterogeneous picture of the folded ensemble for some FnIII domains of tenascin, which may carry important implications for the mechanical functions of tenascins in vivo.

Computer Simulation↗

PubMed1981

Spectroscopic study of the conformations of proline-containing oligopeptides in the crystalline state and in solution.

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d6 solution, using Raman and n.m.r. spectroscopy. The amide I and III bands in the Raman spectra of the crystalline forms indicate the presence of the type I beta-bend conformation in ZGPLG and ZGPLGP, but not in ZGP and ZGPL. This result is in agreement with X-ray data on these molecules. The Raman spectra of these peptides in solution indicate that more than one conformation is present, i.e. that the beta-bend structure of the solid form of ZGPLG and ZGPLGP is destabilized by DMSO-d6. 13C and 1H n.m.r. data also demonstrate the presence of more than one conformation in ZGP, ZGPL, ZGPLG and ZGPLGP in DMSO-d6 solution. These isomers differ in their conformation (cis and trans) about their Gly-Pro peptide bonds and possibly about the c alpha-C' bond of the C-terminal proline in ZGPLGP. For ZGP, ZGPL, ZGPLG and ZGPLGP, the ensemble of conformations in DMSO-d6 includes C5 and C7 hydrogen-bonded rings; in addition, ZGPLGP may contain a small percentage of a beta-bend conformation (at Pro2-Leu3) with trans peptides in both Gly-Pro moieties.

Crystallization↗

PubMed2002

Molecular dynamics simulations of alpha2 --> 8-linked disialoside: conformational analysis and implications for binding to proteins.

Computational methods have played a key role in elucidating the various three-dimensional structures of oligosaccharides. Such structural information, together with other experimental data, leads to a better understanding of the role of oligosaccharide in various biological processes. The disialoside Neu5Ac-alpha2-->8-Neu5Ac appears as the terminal glycan in glycoproteins and glycolipids, and is known to play an important role in various events of cellular communication. Neurotoxins such as botulinum and tetanus require Neu5Ac-alpha2 --> 8-Neu5Ac for infecting the host. Glycoconjugates containing this disialoside and the enzymes catalyzing their biosynthesis are also regulated during cell growth, development, and differentiation. Unlike other biologically relevant disaccharides that have only two linkage bonds, the alpha2-->8-linked disialoside has four: C2-O, O-C8', C8'-C7', and C7'-C6'. The present report describes the results from nine 1 ns MD simulations of alpha2-->8-linked disialoside (Neu5Ac-alpha2-->8-Neu5Ac); simulations were run using GROMOS96 by explicitly considering the solvent molecules. Conformations around the O-C8' bond are restricted to the +sc/+ap regions due to stereochemical reasons. In contrast, conformations around the C2-O and C8'-C7' bonds were found to be largely unrestricted and all the three staggered regions are accessible. The conformations around the C7'-C6' bond were found to be in either the -sc or the anti region. These results are in excellent agreement with the available NMR and potential energy calculation studies. Overall, the disaccharide is flexible and adopts mainly two ensembles of conformations differing in the conformation around the C7'-C6' bond. The flexibility associated with this disaccharide allows for better optimization of intermolecular contacts while binding to proteins and this may partially compensate for the loss of conformational entropy that may be incurred due to disaccharide's flexibility.

Algorithms↗

PubMed2003

Structure and energetics of an allele-specific genetic interaction between dnaJ and dnaK: correlation of nuclear magnetic resonance chemical shift perturbations in the J-domain of Hsp40/DnaJ with binding affinity for the ATPase domain of Hsp70/DnaK.

The molecular chaperone machine composed of Escherichia coli Hsp70/DnaK and Hsp40/DnaJ binds and releases client proteins in cycles of ATP-dependent protein folding, membrane translocation, disassembly, and degradation. The J-domain of DnaJ simultaneously stimulates ATP hydrolysis in the ATPase domain and capture of the client protein in the peptide-binding domain of DnaK. ATP-dependent binding of DnaJ to DnaK mimics DnaJ-dependent capture of a client protein. The dnaJ mutation that replaces aspartate-35 with asparagine (D35N) in the J-domain causes a defect in binding of DnaJ to DnaK. The dnaK mutation that replaces arginine-167 with alanine (R167A) in the ATPase domain of DnaK(R167A) restores binding of DnaJ(D35N). This genetic interaction was said to be allele-specific because wild-type DnaJ does not bind to DnaK(R167A). The J-domain of DnaJ binds to the ATPase domain of DnaK in its capacity as modulator of DnaK ATPase activity and conformational behavior. Surprisingly, the mutations affect the domainwise interaction in an almost opposite manner. D35N increases the affinity of the J-domain for the ATPase domain. R167A has no affect on the affinity of the ATPase domain for the D35N mutant J-domain, but it reduces the affinity for the wild-type J-domain. Previous amide ((1)H, (15)N) NMR chemical shift perturbation mapping in the J-domain suggested that the ATPase domain binds to J-domain helix II and the flanking loops. In the D35N mutant J-domain, chemical shift perturbations include additional effects at amides in the flexible loop II-III and helix III, which have been proposed to undergo an induced fit conformational change upon binding to DnaK. The integrated magnitudes of chemical shift perturbations for the various J-domain and ATPase domain pairs correlate with the free energies of binding. Thus, the J-domain structure can be described as a dynamic ensemble of conformations that is constrained by binding to the ATPase domain. J-domain helix II bends upon binding to the ATPase domain. D35N increases helix II bending, but less so in combination with R167A in the ATPase domain. Taken together, the results suggest that D35N overstabilizes an induced fit conformational change in loop II-III and helix III that is necessary for the J-domain to couple ATP hydrolysis with a conformational change in DnaK, and R167A destabilizes the induced conformation. Conclusions from this work have implications for understanding mechanisms of protein-protein interaction that are involved in allosteric regulation and genetic suppression.

Adenosine Triphosphatases↗

PubMed2026

Dynamic Protein Structure Paradox: An Integrative Framework for Endpoint-Conditioned Evidentiary Sufficiency in Structure-to-Function Claims.

Accurate coordinates for a represented protein state do not, by themselves, establish activity or any other condition-specific function. This article defines the Dynamic Protein Structure Paradox (DPSP) as the apparent conflict between structural accuracy and functional underdetermination and develops it as an integrative evidentiary assessment framework rather than a new theory or paradigm. The underlying problem has been longstanding, since structural genomics, function annotation, allostery, and disorder research each established that fold does not determine function and that function does not determine fold. DPSP consolidates those results into one endpoint-conditioned rule. Once a measurable endpoint is defined, it assesses four coupled dimensions: relevant-state completeness, context completeness, ensemble or kinetic dependence, and chemical dependence. A rubric rates each dimension as adequate, uncertain, or missing, and a materiality test determines which gaps influence the stated decision. The outcome is one of three mutually exclusive modes of utilization: geometry-led, conditional, or function-measured. The deliverable is a concise evidence statement delineating what the structure supports, which decisive variable remains unmeasured, and what corroboration is necessary. DPSP complements, rather than replaces, existing structural, ensemble, and computational approaches. The framework remains unvalidated, its thresholds are provisional, and the studies necessary to confirm or refute it are specified.

Proteins↗

PubMed1991

A new method for building protein conformations from sequence alignments with homologues of known structure.

We describe a largely automatic procedure for building protein structures from sequence alignments with homologues of known structure. This procedure uses simple rules by which multiple sequence alignments can be translated into distance and chirality constraints, which are then used as input for distance geometry calculations. By this means one obtains an ensemble of conformations for the unknown structure that are compatible with the rules employed, and the differences among these conformations provide an indication of the reliability of the structure prediction. The overall approach is demonstrated here by applying it to several Kazal-type trypsin inhibitors, for which experimentally determined structures are available. On the basis of our experience with these test problems, we have further predicted the conformation of the human pancreatic secretory trypsin inhibitor, for which no experimentally determined structure is presently available.

Algorithms↗

PubMed2006

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics.

The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.

Animals↗

PubMed2000

Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation.

Nuclear receptors are ligand-dependent transcription factors that are mediators of the action of lipophilic hormones and other endogenous ligands and are the targets of drugs useful in a variety of therapeutic areas. Peroxisome proliferator-activated receptor (PPAR)gamma is a nuclear receptor that, acting as a heterodimer with RXR, mediates a variety of cellular effects including adipocyte-differentiation. Due to its role in modulating insulin sensitivity, it is the target of therapeutically active anti-diabetic agents such as rosiglitazone. We have assigned the chemical shifts of the backbone atoms of the 32 kDa ligand-binding domain of PPARgamma in the presence of bound rosiglitazone. Three-dimensional HNCO spectra of the apo ligand-binding domain (LBD) have less than half the expected number of cross-peaks. The missing cross-peaks are restored upon binding strong agonists such as rosiglitazone. The NMR results indicate that the apo-LBD of PPARgamma is in a conformationally mobile state, and that agonist binding is associated with a marked stabilization of the conformation. Mapping the missing peaks to the 3D X-ray crystallographic structure indicates the region of mobility is extensive and includes the ligand-binding region and the cofactor-binding site. This leads to the conclusion that activation of this nuclear receptor is a result of a population shift of a dynamic ensemble of conformations, rather than a two-state switch from an inactive to an active conformation. Our results have important implications for the mechanisms by which antagonists, partial agonists, and agonists of nuclear receptor function operate.

Amino Acid Sequence↗

PubMed2004

Correlated C-C and C-O bond conformations in saccharide hydroxymethyl groups: parametrization and application of redundant 1H-1H, 13C-1H, and 13C-13C NMR J-couplings.

Methyl alpha- and beta-pyranosides of d-glucose and d-galactose 1-4 were prepared containing single sites of (13)C-enrichment at C4, C5, and C6 (12 compounds), and (1)H and (13)C[(1)H] NMR spectra were obtained to determine a complete set of J-couplings ((1)J, (2)J, and (3)J) involving the labeled carbon and nearby protons and carbons within the exocyclic hydroxymethyl group (CH(2)OH) of each compound. In parallel theoretical studies, the dependencies of (1)J, (2)J, and (3)J involving (1)H and (13)C on the C5-C6 (omega) and C6-O6 (theta;) torsion angles in aldohexopyranoside model compounds were computed using density functional theory (DFT) and a special basis set designed to reliably recover the Fermi contact contribution to the coupling. Complete hypersurfaces for (1)J(C5,C6), (2)J(C5,H6)(R), (2)J(C5,H6)(S), (2)J(C6,H5), (2)J(C4,C6), (3)J(C4,H6)(R), (3)J(C4,H6)(S), and (3)J(C6,H4), as well as (2)J(H6)(R)(,H6)(S), (3)J(H5,H6)(R), and (3)J(H5,H6)(S), were obtained and used to parametrize new equations correlating these couplings to omega and/or theta;. DFT-computed couplings were also tested for accuracy by measuring J-couplings in (13)C-labeled 4,6-O-ethylidene derivatives of d-glucose and d-galactose in which values of omega and theta; were constrained. Using a new computer program, Chymesa, designed to utilize multiple J-couplings sensitive to exocyclic CH(2)OH conformation, the ensemble of experimental couplings observed in 1-4 were analyzed to yield preferred rotamer populations about omega and theta;. Importantly, due to the sensitivity of some couplings, most notably (2)J(H6)(R)(,H6)(S), (2)J(C5,H6)(R), and (2)J(C5,H6)(S), to both omega and theta;, unique information on correlated conformation about both torsion angles was obtained. The latter treatment represents a means of evaluating correlated conformation in 1,6-linked oligosaccharides, since psi and theta; are redundant in these linkages. In the latter regard, multiple, redundant scalar couplings originating from both sides of the glycosidic linkage can be used collectively to evaluate conformational correlations between psi/theta; and C5-C6 bond rotamers.

Carbohydrate Conformation↗

PubMedDate not supplied

Non-uniform triple helical structure in chick skin type I collagen on thermal denaturation: Raman spectroscopic study.

The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 degrees C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.

Animals↗

PubMed1993

Distribution of the iron-heme displacement as resulting from myoglobin conformational substrates: an AOM approach to the interpretation of the EPR spectra.

The electron paramagnetic resonance (EPR) low-temperature spectra of high spin ferric myoglobin samples in different solvent composition have been analyzed in terms of a distribution of the energy differences delta 1 and delta 2 for the iron low-lying electronic states. The widths of these distributions, which are found to be dependent on the solvent composition, have been correlated to the presence of a frozen ensemble of conformational substrates. A dedicated analysis based on the angular overlap method (AOM) has allowed us to work out a quantitative relationship between the delta 1 and delta 2 distributions and the spread of the iron-heme displacement; this being a structural parameter relevant for the biological functionality of the protein. The observed dependence of the iron-heme displacement distribution on the solvent composition is discussed.

Animals↗

PubMed2006

Singular value decomposition of torsional angles of analogs of the dopamine reuptake inhibitor GBR 12909.

Analysis of large, flexible molecules, such as the dopamine reuptake inhibitor GBR 12909 (1), is complicated by the fact that they can take on a wide range of closely related conformations. The first step in the analysis is to classify the conformers into groups. Here, Singular Value Decomposition (SVD) was used to group conformations of GBR 12909 analogs by the similarity of their nonring torsional angles. The significance of the present work, the first application of SVD to the analysis of very flexible molecules, lies in the development of a novel scaling technique for circular data and in the grouping of molecular conformations using a technique that is independent of molecular alignment. Over 700 conformers each of a piperazine (2) and piperidine (3) analog of 1 were studied. Analysis of the score and loading plots showed that the conformers of 2 separate into three large groups due to torsional angles on the naphthalene side of the molecule, whereas those of 3 separate into nine groups due to torsional angles on the bisphenyl side of the molecule. These differences are due to nitrogen inversion at the unprotonated piperazinyl nitrogen of 2, which results in a different ensemble of conformers than those of 3, where no inversion is possible at the corresponding piperidinyl carbon.

Computer Simulation↗

PubMed1998

Efficient dynamics in the space of contact maps.

BACKGROUND: Two problems are of major importance in protein fold prediction: how to generate plausible conformations, and how to choose an energy function to identify the native state. Contact maps are a simple representation of protein structure and offer a promising framework to address these two issues. RESULTS: In this work we develop Monte Carlo dynamics in contact map space. The procedure is divided into four steps: non-local dynamics, in which large-scale "cluster" moves are performed (clusters are in approximate correspondence with secondary structure elements); local dynamics, in which secondary structure location is optimized; reconstruction, in which the physicality of the contact map is restored; and refinement, which consists of a further Monte Carlo energy minimization in real space. We demonstrate that such a dynamical procedure is effective in producing uncorrelated low-energy states. CONCLUSIONS: The procedure introduced in this paper very effectively generates a representative ensemble of conformations. We are able to show that existing sets of pairwise contact energy parameters are not suitable to single out the native state within this ensemble. The remaining outstanding issue in protein folding is to find an energy function that can discriminate the native state from decoys.

Models, Chemical↗

PubMed1981

Theoretical conformational analysis of enkephalin analogues related to fluorescence and n.m.r. measurements in aqueous solution.

The properties related to non-radiative energy transfer of a number of enkephalin analogues with tryptophan substituted for phenylalanine in position 4 and n.m.r. 3JNH-C alpha H coupling constants of corresponding [Phe4]-enkephalin analogues are being derived from semi-empirical conformational energy. The molecules considered contain a glycyl, a D-alanyl or an L-alanyl as second residue; two of the compounds are N-methylated at position 4 or 5. The [Trp4]-enkephalin analogues and the corresponding [Phe4]-enkephalin analogues display nearly parallel affinities in the opiate receptor binding assay (Schiller et al. (1). The comparison of computed and experimental properties shows that an ensemble of conformers is a satisfactory representation of the state of these molecules in water.

Endorphins↗

PubMed2006

Comparison of different torsion angle approaches for NMR structure determination.

A new procedure for NMR structure determination, based on the Internal Coordinate Molecular Dynamics (ICMD) approach, is presented. The method finds biopolymer conformations that satisfy usual NMR-derived restraints by using high temperature dynamics in torsion angle space. A variable target function algorithm gradually increases the number of NOE-based restraints applied, with the treatment of ambiguous and floating restraints included. This soft procedure allows combining artificially high temperature with a general purpose force-field including Coulombic and Lennard-Jones non-bonded interactions, which improves the quality of the ensemble of conformations obtained in the gas-phase. The new method is compared to existing algorithms by using the structures of eight ribosomal proteins earlier obtained with state-of-the-art procedures and included into the RECOORD database [Nederveen, A., Doreleijers, J., Vranken, W., Miller, Z., Spronk, C., Nabuurs, S., Guntert, P., Livny, M., Markley, M., Nilges, M., Ulrich, E., Kaptein, R. and Bonvin, A.M. (2005) Proteins, 59, 662-672]. For the majority of tested proteins, the ICMD algorithm shows similar convergence and somewhat better quality Z scores for the phi, psi distributions. The new method is more computationally demanding although the overall load is reasonable.

Algorithms↗

PubMed1993

Interaction of calcium channel antagonists with calcium: structural studies on verapamil and its Ca2+ complex.

The conformation of the calcium channel antagonist verapamil has been determined in acetonitrile, in the absence and presence of Ca2+, using two-dimensional 1H-NMR and molecular modeling techniques. Interproton connectivities in the drug molecule were identified from the observed NOESY cross peaks and interproton distances were estimated from the magnitudes of the volume integrals of the cross peaks. The molecular modeling program utilized the Monte Carlo simulation to generate a random ensemble of conformers complying with the NOESY-derived distance constraints. The energies of these conformers were subsequently computed. The minimum-energy structure of the free drug obtained in this manner exhibited some significant differences from the structure of verapamil determined by X-ray crystallography. In particular, the torsional angles in the middle region of the molecule containing the aliphatic "backbone" were such that the two aromatic rings at either end of the drug molecules were moved farther apart from each other in solution than in the crystal structure. The nearly perpendicular orientation of the aromatic rings seen in the crystal was, however, maintained in the solution structure as well. The addition of Ca2+ to a solution of verapamil in acetonitrile caused marked changes in the difference absorbance of the drug in the 200-300-nm region and in many of its 1H-NMR resonances. The changes were most significant up to a mole ratio of about 0.5 Ca2+:drug. Analysis of the binding data at 25 degrees C showed the presence of both 2:1 and 1:1 drug:Ca2+ complexes in equilibrium, the former "sandwich" complex being dominant at the lower cation concentrations with an estimated dissociation constant of about 300 microM. All of the NOESY cross peaks of the free drug remained on addition of 0.5 mol ratio of Ca2+ to verapamil in deuterated acetonitrile and only two new connectivities were observed. Using the interproton distances calculated from these NOESY data, molecular modeling of the 2:1 drug:Ca2+ complex was carried out to yield the minimum-energy conformer. In this conformer, Ca2+ was coordinated to two methoxy oxygens from each of the two drug molecules. The implications of the verapamil-Ca2+ interaction are discussed in terms of available experimental data on the binding of verapamil to the dihydropyridine-sensitive channel and in terms of a hypothesis on the formation of a drug-Ca(2+)-receptor complex in the lipid bilayer environment.

Binding Sites↗

PubMed2001

Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering.

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and fluorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-defined states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-defined states.

Calorimetry, Differential Scanning↗