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Results for “conformational ensemble”

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PubMed2003

Hydrogen exchange in a large 29 kD protein and characterization of molten globule aggregation by NMR.

The nature of denatured ensembles of the enzyme human carbonic anhydrase (HCA) has been extensively studied by various methods in the past. The protein constitutes an interesting model for folding studies that does not unfold by a simple two-state transition, instead a molten globule intermediate is highly populated at 1.5 M GuHCl. In this work, NMR and H/D exchange studies have been conducted on one of the isozymes, HCA I. The H/D exchange studies, which were enabled by the previously obtained resonance assignment of HCA I, have been used to identify unfolded forms that are accessible from the native state. In addition, the GuHCl-induced unfolded states of HCA I have also been characterized by NMR at GuHCl concentrations in the 0-5 M range. The most important findings in this work are as follows: (1) Amide protons located in the center of the beta-sheet require global unfolding events for efficient H/D exchange. (2) The molten globule and the native state give similar protection against H/D exchange for all of the observable amide protons (i.e., water seems not to efficiently penetrate the interior of the molten globule). (3) At high protein concentrations, the molten globule can form large aggregates, which are not detectable by solution-state NMR methods. (4) The unfolded state (U), present at GuHCl concentrations above 2 M, is composed of an ensemble of conformations having residual structures with different stabilities.

Amides↗

PubMed2006

Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by 19F and 1H NMR.

The isolated N-terminal SH3 domain of the Drosophila signal transduction protein Drk (drkN SH3) is a useful model for the study of residual structure and fluctuating structure in disordered proteins since it exists in slow exchange between a folded (Fexch) and compact unfolded (Uexch) state in roughly equal proportions under nondenaturing conditions. The single tryptophan residue, Trp36, is believed to play a key role in forming a non-native hydrophobic cluster in the Uexch state, with a number of long-range nuclear Overhauser contacts (NOEs) observed primarily to the indole proton. Substitution of Trp36 for 5-fluoro-Trp36 resulted in a substantial shift in the equilibrium to favor the Fexch state. A variety of 19F NMR measurements were performed to investigate the degree of solvent exposure and hydrophobicity associated with the 5-fluoro position in both the Fexch and Uexch states. Ambient T1 measurements and H2O/D2O solvent isotope effects indicated extensive protein contacts to the 5-fluoro position in the Fexch state and greater solvent exposure in the Uexch state. This was corroborated by the measurements of paramagnetic effects (chemical shift perturbations and T1 relaxation enhancement) from dissolved oxygen at a partial pressure of 20 atm. In contrast, paramagnetic effects from dissolved oxygen revealed less solvent exposure to the indole proton of Trp36 in the Uexch state than that observed for the Fexch state, consistent with the model in which Trp36 indole belongs to a non-native cluster. Thus, although the Uexch state may be described as a dynamically interconverting ensemble of conformers, there appears to be significant asymmetry in the environment of the indole group and the six-membered ring or backbone of Trp36. This implied lack of averaging of a side chain position is in contrast to the general view of fluctuating side chains within disordered states.

Fluorine↗

PubMed1998

Solution structure of an intramolecular DNA triplex linked by hexakis(ethylene glycol) units: d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT).

A DNA molecule was designed and synthesized with three octanucleotide stretches linked by two hexakis(ethylene glycol) chains to form an intramolecular triplex in solution. The structural data obtained from a series of NMR NOESY spectra yielded interproton distances, and COSY experiments provided dihedral angle information for analysis of deoxyribose ring pucker. Using distance geometry followed by simulated annealing with restrained molecular dynamics and relaxation matrix refinement, a well-refined ensemble of conformations was calculated. Although some NOE cross-peaks involving protons of the hexakis(ethylene glycol) linker could be identified, most could not be assigned and the conformations of the linkers were not determined. The deoxyribose conformations are predominantly of the S type, except for the protonated cytosine residues in the third strand which show hybrid N and S character. Overall, the duplex part of the molecule resembles a B-DNA double helix with the third strand bound in its major groove by Hoogsteen hydrogen bonds. This structure provides a basis for comparison with triplexes containing noncanonical or nonnatural nucleotides.

Carbohydrate Conformation↗

PubMed1999

Structural transitions in the protein L denatured state ensemble.

We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.

Amino Acid Sequence↗

PubMed2004

On the interpretation of residual dipolar couplings as reporters of molecular dynamics.

The analysis of residual dipolar couplings from an ensemble of conformations to extract molecular dynamics is intricate. The very mechanism that is necessary to perturb overall molecular tumbling to generate nonvanishing residual dipolar couplings gives rise to convoluted data. The measured values are essentially weighted averages over conformations. However, the weights are not simply the populations of conformations. Consequently, the observed order parameter is not exactly the true measure of motion. In the case of paramagnetic alignment, the apparent order parameter is expected to depend on the number of torsions that separate the locus of interest from the paramagnetic site. In the case of alignment due to steric obstruction, the uneven selection of conformations by their differing Saupe order matrices leads to a bias in the residual dipolar couplings-probed molecular dynamics.

Models, Molecular↗

PubMed2000

Binding of a diverse set of ligands to avidin and streptavidin: an accurate quantitative prediction of their relative affinities by a combination of molecular mechanics and continuum solvent models.

We report calculations of free energies of binding, DeltaG(bind), between a diverse set of nine ligands and avidin as well as between a peptide and streptavidin using the recently developed MM/PBSA approach. This method makes use of a molecular dynamics simulation of the ligand-protein complex to generate a thermally averaged ensemble of conformations of the molecules that are involved in the complex formation. Based on this set of structures, a free energy of binding is calculated using molecular mechanical and continuum solvent energies as well as including estimates of the nonpolar solvation free energy and solute entropy. We compare in our simulations different classes of ligands, involving biotin derivatives, the dye 2-(4'-hydroxyazobenzene)benzoic acid (HABA), and a cyclic hexapeptide, which cover a large range of binding free energies from -5 to -20 kcal/mol. Our calculations are able to reproduce experimental DeltaG(bind) values with a very good correlation coefficient of r(2) = 0.92. This agreement is considerably better than the results obtained with an alternate approach, the linear interaction energy approximation, for this system (r(2) = 0.55).

Avidin↗

PubMed2006

Protein intrinsic disorder and human papillomaviruses: increased amount of disorder in E6 and E7 oncoproteins from high risk HPVs.

It is recognized now that many functional proteins or their long segments are devoid of stable secondary and/or tertiary structure and exist instead as very dynamic ensembles of conformations. They are known by different names including natively unfolded, intrinsically disordered, intrinsically unstructured, rheomorphic, pliable, and different combinations thereof. Many important functions and activities have been associated with these intrinsically disordered proteins (IDPs), including molecular recognition, signaling, and regulation. It is also believed that disorder of these proteins allows function to be readily modified through phosphorylation, acetylation, ubiquitination, hydroxylation, and proteolysis. Bioinformatics analysis revealed that IDPs comprise a large fraction of different proteomes. Furthermore, it is established that the intrinsic disorder is relatively abundant among cancer-related and other disease-related proteins and IDPs play a number of key roles in oncogenesis. There are more than 100 different types of human papillomaviruses (HPVs), which are the causative agents of benign papillomas/warts, and cofactors in the development of carcinomas of the genital tract, head and neck, and epidermis. With respect to their association with cancer, HPVs are grouped into two classes, known as low (e.g., HPV-6 and HPV-11) and high-risk (e.g., HPV-16 and HPV-18) types. The entire proteome of HPV includes six nonstructural proteins [E1, E2, E4, E5, E6, and E7 (the latter two are known to function as oncoproteins in the high-risk HPVs)] and two structural proteins (L1 and L2). To understand whether intrinsic disorder plays a role in the oncogenic potential of different HPV types, we have performed a detailed bioinformatics analysis of proteomes of high-risk and low-risk HPVs with the major focus on E6 and E7 oncoproteins. The results of this analysis are consistent with the conclusion that high-risk HPVs are characterized by the increased amount of intrinsic disorder in transforming proteins E6 and E7.

Algorithms↗

PubMed2004

Alignment of chain-like molecules.

The steric obstruction model, that describes the enhanced alignment of folded proteins by anisotropic medium, is extended to account for the residual dipolar couplings of chain-like polypeptides. The average alignment of each chain segment is calculated from an ensemble of conformations represented by a spatial probability distribution. The segmental alignment depends on chain length, flexibility and segment's position in the chain. Residual dipolar couplings in turn depend on internuclear vector directions within each fragment. The results of calculations and simulations explain salient features of the experimental data. With this insight residual dipolar couplings can be interpreted to assess the degree of denaturation, local structures and spatial organization of weakly structured proteins.

Data Interpretation, Statistical↗

PubMed2004

Importance of chirality and reduced flexibility of protein side chains: a study with square and tetrahedral lattice models.

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.

Computer Simulation↗

PubMed2004

Theoretical calculations of infrared absorption, vibrational circular dichroism, and two-dimensional vibrational spectra of acetylproline in liquids water and chloroform.

Infrared absorption, vibrational circular dichroism, and two-dimensional infrared pump-probe and photon echo spectra of acetylproline solutions are theoretically calculated and directly compared with experiments. In order to quantitatively determine interpeptide interaction-induced amide I mode frequency shifts, high-level quantum chemistry calculations were performed. The solvatochromic amide I mode frequency shift and fluctuation were taken into account by carrying out molecular dynamics simulations of acetylproline dissolved in liquids water and chloroform and by using the extrapolation method developed recently. We first studied correlation time scales of the two amide I vibrational frequency fluctuations, cross correlation between the two fluctuating local mode frequencies, ensemble averaged conformations of the acetylproline molecule in liquids water and chloroform. The corresponding conformations of the acetylproline in liquids water and chloroform are close to the ideal 3(10) helix and the C(7) structure, respectively. A few methods proposed to determine the angle between the two transition dipoles associated with the amide I vibrations were tested and their limitations are discussed.

Journal Article↗

PubMed2005

Mesoscale model of polymer melt structure: self-consistent mapping of molecular correlations to coarse-grained potentials.

Development and application of coarse-graining methods to condensed phases of macromolecules is an active area of research. Multiscale modeling of polymeric systems using coarse-graining methods presents unique challenges. Here we apply a coarse-graining method that self-consistently maps structural correlations from detailed molecular dynamics (MD) simulations of alkane oligomers onto coarse-grained potentials using a combination of MD and inverse Monte Carlo methods. Once derived, the coarse-grained potentials allow computationally efficient sampling of ensemble of conformations of significantly longer polyethylene chains. Conformational properties derived from coarse-grained simulations are in excellent agreement with experiments. The level of coarse graining provides a control over the balance of computational efficiency and retention of chemical identity of the underlying polymeric system. Challenges to extension and application of this and similar structure-based coarse-graining methods to model dynamics and phase behavior in polymeric systems are briefly discussed.

Journal Article↗

PubMed2006

Two-dimensional electronic spectra of symmetric dimers: Intermolecular coupling and conformational states.

We study the information content of two-dimensional (2D) electronic photon-echo (PE) spectra, with special emphasis on their potential to distinguish, for waiting times T=0, between different conformations of electronically coupled symmetric dimers. The analysis is performed on the basis of an analytical formula for the frequency-domain 2D PE signal. The symmetric dimers are modeled in terms of two identical, energy-degenerate, excitonically coupled pairs of electronic states in the site representation. The spectra of conformationally weighted ensembles, composed of either two or four dimers, are compared with their one-dimensional linear absorption counterparts. In order to provide a realistic coupling pattern for the ensemble consisting of four dimers, excitonic couplings are estimated on the basis of optimized geometries and site-transition dipole moments, calculated by standard semiempirical methods for the bridged bithiophene structure 1,2-bithiophene-2-yl-ethane-1,2-dion (T2[CO]2). In the framework of our model, the highly readable 2D PE spectra can unambiguously identify spectral doublets, by relating peak heights and positions with mutual orientations of site-localized transition dipoles.

Journal Article↗

PubMed2006

Absence of reptation in the high-temperature folding of the trpzip2 beta-hairpin peptide.

We have carried out extensive all atom explicit solvent simulations of the high-temperature folding and unfolding of the trpzip2 beta-hairpin peptide and examined the resulting trajectories for evidence of folding via a reptation mechanism. Over 300 microcanonical simulations of 10 ns each were initiated from a Boltzmann ensemble of conformations at 425 K. Though we observed numerous folding and unfolding events, no evidence of reptation was found. The diffusional dynamics of the peptide are orders of magnitude faster than any observed reptation-like motion. Our data suggest that the dominant mechanisms for beta-hairpin folding under these conditions are hydrophobic collapse and turn formation, and that rearrangements occur via significant expansion of the polypeptide chain.

Chemistry, Physical↗

PubMed2006

A kinetic theory of tertiary contact formation coupled to the helix-coil transition in polypeptides.

The framework model and the hydrophobic collapse model represent two canonical descriptions of the protein folding process. The first places primary reliance on the short-range interactions of secondary structure and the second assigns greater importance to the long-range interactions of tertiary structure. The availability of increasingly detailed information about the folding mechanisms of diverse proteins suggests that both are important and the folding mechanism of most proteins utilizes different combinations of such interactions. A prior report described the XHC model, an extended helix-coil theory, which treats the mutual stabilization of secondary and tertiary structure in simple alpha-helical proteins at equilibrium. In this study, a kinetic scheme describing tertiary contact formation has been developed which relaxes to the XHC equilibrium model. The relaxation is governed by the relative stabilities of the equilibrium states and an additional factor which represents an activation energy for formation of a tertiary contact. The model can be used to simulate time-dependent properties of the ensemble of conformations during the entire folding process, and the resulting predictions are applicable to a range of experimental methods. This XHC kinetic model enables investigation of the relative influence of secondary and tertiary interactions on folding mechanisms.

Algorithms↗

PubMed2005

Altering the interaction between sigma70 and RNA polymerase generates complexes with distinct transcription-elongation properties.

We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from sigma70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between sigma70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of sigma70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and sigma70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase.

DNA-Directed RNA Polymerases↗

PubMed2002

Molecular dynamics simulations of protein folding from the transition state.

Putative transition-state ensemble (TSE) conformations of src SH3 were identified by monitoring the deviation from the experimental phi values along molecular dynamics (MD) simulations of unfolding. Sixty MD trajectories (for a total of about 7 micros) were then started from the putative TSE. About one-half of the 60 runs reached the folded state while unfolding was observed in the remaining half of the runs. This result validates phi-value analysis as an approach to obtain structural information on the transition state. It also demonstrates that an atomic resolution description of the TSE can be extracted from MD simulations. All conformations in the TSE have the central three-stranded beta-sheet formed in agreement with experimental data. An elongation of strand beta 2 as well as non-native side-chain interactions between the diverging turn and the distal hairpin are observed. The simulation results indicate that the tight packing of the side chains between the diverging turn and the distal hairpin is a necessary condition for rapid folding. Contacts between residues in the most structured element of the TSE, the central beta-sheet, are kinetically more important than those between the N- and C-terminal strands.

Computer Simulation↗

PubMed1998

The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation.

Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescent N-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and 15N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is alpha-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.

Amino Acid Sequence↗

PubMed2000

Progressive stabilization of intermediate and transition states in protein folding reactions by introducing surface hydrophobic residues.

It can be argued from the principle of solvent exclusion that the introduction of hydrophobic residues onto the surface of a protein will not destabilize the folded state because the nonpolar side chain will be at least as exposed in the unfolded state as it is when the protein chain is folded. A comparison of the folding pathway of wild type and 11 site-directed mutants of CD2.d1 shows this to be true. In fact, owing to partial burial of nonpolar groups as folding proceeds, we find that the rapidly formed intermediate state and, to a greater extent, the transition state are generally stabilized by hydrophobic surface mutations. This effect is slightly moderated in the folded state presumably by the perturbation of van der Waals' contacts and/or local electrostatic interactions that have a greater influence in this fully compact structure. The fact that in all but one case we find that stabilization of the rapidly collapsed intermediate is accompanied by a faster acquisition of the folded state refutes the argument that I states are generally "off pathway" conformations or ensembles that lead to the inhibition of otherwise more rapid folding trajectories.

Amino Acid Substitution↗