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PubMed2000

Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide.

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N-oxide (TMAO). The structure of CspA has five strands of beta-sheet. Strands beta1-beta4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all-or-none global unfolding mechanism. By contrast, the protection of amide protons from strand beta5 is too weak to measure in water. Strand beta5 is hydrogen bonded to strands beta3 and beta4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands beta1-beta4 but fail to account for the weak protection in strand beta5. The most conspicuous feature of strand beta5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands beta1-beta4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand beta5 and the loops connecting the beta-strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen-bonding interactions in the native structure.

Bacterial Proteins↗
PubMed2003

Protein dynamics from solution NMR: theory and applications.

Solution nuclear magnetic resonance (NMR) spectroscopy is unique in its ability to elucidate the details of atomic-level structural and dynamical properties of biological macromolecules under native-like conditions. Recent advances in NMR techniques and protein sample preparation now allow comprehensive investigation of protein dynamics over timescales ranging 14 orders of magnitude at nearly every atomic site. Thus, solution NMR is poised to reveal aspects of the physico-chemical properties that govern the ensemble distribution of protein conformers and the dynamics of their interconversion. We review these advances as well as their recent application to the study of proteins.

Carbon↗
PubMed2002

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor.

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R) that confers agonist selectivity for Arg(8) in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L(296), N(297), and R(298). A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Amino Acid Sequence↗
PubMed2004

Designing generalized statistical ensembles for numerical simulations of biopolymers.

Conformational properties of polymers, such as average dihedral angles or molecular alpha-helicity, display a rather weak dependence on the detailed arrangement of the elementary constituents (atoms). We propose a computer simulation method to explore the polymer phase space using a variant of the standard multicanonical method, in which the density of states associated to suitably chosen configurational variables is considered in place of the standard energy density of states. This configurational density of states is used in the Metropolis acceptance/rejection test when configurations are generated with the help of a hybrid Monte Carlo algorithm. The resulting configurational probability distribution is then modulated by exponential factors derived from the general principle of the maximal constrained entropy by requiring that certain average configurational quantities take preassigned (possibly temperature dependent) values. Thermal averages of other configurational quantities can be computed by using the probability distributions obtained in this way. Moments of the energy distribution require an extra canonical sampling of the system phase space at the desired temperature, in order to locally thermalize the configurational degrees of freedom. As an application of these ideas we present the study of the structural properties of two simple models: a bead-and-spring model of polyethylene with independent hindered torsions and an all-atom model of alanine and glycine oligomers with 12 amino acids in vacuum.

Biopolymers↗
PubMed2005

[Aladan3]TIPP: a fluorescent delta-opioid antagonist with high delta-receptor binding affinity and delta selectivity.

Fluorescent analogues of the potent and highly selective delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid beta-(6'-dimethylamino-2'-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald3 analogues of TIPP and TIP all retained delta-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K(e) value of 2.03 nM in the mouse vas deferens assay and five times higher delta vs. mu selectivity (K(i)mu/K(i)delta = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) delta-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.

2-Naphthylamine↗
PubMed2003

Solution structure, dynamics, and thermodynamics of the native state ensemble of the Sem-5 C-terminal SH3 domain.

Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.

Algorithms↗
PubMed2004

Free energies of binding of polychlorinated biphenyls to the estrogen receptor from a single simulation.

Relative free energies of binding to the ligand-binding domain of the estrogen receptor have been calculated for a series of 17 hydroxylated polychlorinated biphenyls. Because traditional thermodynamic integration or perturbation approaches are hardly feasible for these numbers of compounds, the one-step perturbation approach is applied and is shown to yield accurate results based on only two 2-ns molecular dynamics simulations of an unphysical, judiciously chosen, reference state. The mean absolute difference between the calculated and experimental binding free energies for the 17 compounds is 3.4 kJ/mol, which illustrates the accuracy of the GROMOS biomolecular force field used. Excluding the three largest ligands from the comparison reduces the deviation to 2.0 kJ/mol (i.e., < k(B)T). Apart from the relative free energy, structural information about the binding mode and binding orientation for every compound can also be extracted from the simulation, showing that a ligand bound to its receptor cannot be represented by a single conformation, but it samples an ensemble of different orientations.

Carbon↗
PubMed2001

Conformation spaces of proteins.

We report a simple method for measuring the accessible conformational space explored by an ensemble of protein structures. The method is useful for diverse ensembles derived from molecular dynamics trajectories, molecular modeling, and molecular structure determinations. It can be used to examine a wide range of time scales. The central tactic we use, which has been previously employed, is to replace the true mechanical degrees of freedom of a molecular system with the conformationally effective degrees of freedom as measured by the root-mean squared cartesian distances among all pairs of conformations. Each protein conformation is treated as a point in a high dimensional euclidean space. In this article, we model this space in a novel way by representing it as an N-dimensional hypercube, describable with only two parameters: the number of dimensions and the edge length. To validate this approach, we provide a number of elementary test cases and then use the N-cube method for measuring the size and shape of conformational space covered by molecular dynamics trajectories spanning 10 orders of magnitude in time. These calculations were performed on a small protein, the villin headpiece subdomain, exploring both the native state and the misfolded/folding regime. Distinct features include single, vibrationally averaged, substate minima on the 0.1-1-ps time scale, thermally averaged conformational states that persist for 1-100 ps and transitions between these local minima on nanosecond time scales. Large-scale refolding modes appear to become uncorrelated on the microsecond time scale. Associated length scales for these events are 0.2 A for the vibrational minima; 0.5 A for the conformational minima; and 1-2 A for the nanosecond events. We find that the conformational space that is dynamically accessible during folding of villin has enough volume for approximately 10(9) minima of the variety that persist for picoseconds. Molecular dynamics trajectories of the native protein and experimentally derived solution ensembles suggest the native state to be composed of approximately 10(2) of these thermally accessible minima. Thus, based on random exploration of accessible folding space alone, protein folding for a small protein is predicted to be a milliseconds time scale event. This time can be compared with the experimental folding time for villin of 10-100 micros. One possible explanation for the 10-100-fold discrepancy is that the slope of the "folding funnel" increases the rate 1-2 orders of magnitude above random exploration of substates.

Algorithms↗
PubMed2006

Stabilizing IkappaBalpha by "consensus" design.

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Amino Acid Sequence↗
PubMed2003

Formation and structure of self-assembled monolayers of alkanethiolates on palladium.

The adsorption of n-alkanethiols onto polycrystalline thin films of palladium containing a strong (111) texture produces well-organized, self-assembled monolayers. The organization of the alkane chains in the monolayer and the nature of the bonding between the palladium and the thiol were studied by contact angle measurements, optical ellipsometry, reflection absorption infrared spectroscopy (RAIRS), and X-ray photoelectron spectroscopy (XPS). The XPS data reveals that a compound palladium-sulfide interphase is present at the surface of the palladium film. The RAIR spectra, ellipsometry data, and wetting properties show that the palladium-sulfide phase is terminated with an organized, methyl-terminated monolayer of alkanethiolates. The local molecular environment of the alkane chains transitions from a conformationally disordered, liquidlike state to a mostly all-trans, crystalline-like structure with increasing chain length (n = 8-26). The intensities and dichroism of the methylene and methyl stretching modes support a model for the average orientation of an ensemble of all-trans-conformer chains with a tilt angle of approximately 14-18 degrees with respect to the surface normal and a twist angle of the CCC plane relative to the tilt plane of approximately 45 degrees. The SAMs are stable in air, although the sulfur present at the surface oxidizes in air over a period of 2-5 days at room temperature. The differences in chain organization between SAMs formed by microcontact printing and by solution deposition are also examined by RAIRS and XPS.

Air↗
PubMed2003

Reintroducing electrostatics into protein X-ray structure refinement: bulk solvent treated as a dielectric continuum.

Structural refinement of proteins involves the minimization of a target function that combines X-ray data with a set of restraints enforcing stereochemistry and packing. Electrostatic interactions are not ordinarily included in the target function, partly because they cannot be calculated reliably without a description of dielectric screening by solvent in the crystal. With the recent development of accurate implicit solvent models to describe this screening, the question arises as to whether a more detailed target function including electrostatic and solvation terms can yield more accurate structures or somewhat different structures of equivalent accuracy. The Generalized Born (GB) model is one such model that describes the solvent as a dielectric continuum, taking into account its heterogeneous distribution within the crystal. It is used here for X-ray refinements of three protein structures with experimental diffraction data to 2.4, 2.9 and 3.2 A, respectively. In each case, a higher resolution structure is available for comparison. The new target function includes stereochemical restraints, van der Waals, Coulomb and solvation interactions, along with the usual X-ray pseudo-energy term, which employs the likelihood estimator of Pannu and Read. Multiple simulated-annealing refinements were performed in torsion-angle space with a conventional target function and the new GB target function, yielding ensembles of refined structures. The new target function yields structures of similar accuracy, as measured by the free R factor, map/model correlations and deviations from the high-resolution structures. About 10% of side-chain conformations differ between the two sets of refinements, in the sense that the two ensembles of conformations do not completely overlap. Over 75% of the differences correspond to surface side chains. For one of the proteins, the GB set has a greater dispersion, indicating that for this case the conventional target function overestimates the true precision. As GB parameterization continues to improve, we expect that this approach will become increasingly useful.

Aspartate-tRNA Ligase↗
PubMed1993

Experimental studies and potential energy calculations of the blocked tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the third loop of short-chain snake venom neurotoxins.

The conformational space of the tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the beta-bend present in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations, resulting in the identification of an ensemble of beta-turn conformations. These results were compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. A random coil conformation of the peptide is indicated in polar hydrogen-bonding solvents. In less polar solvents the peptide backbone assumed a more rigid conformation, as reflected by the existence of at least a type II beta-turn conformation.

Amino Acid Sequence↗
PubMed2003

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase.

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.

Circular Dichroism↗
PubMed2005

Structural function of C-terminal amidation of endomorphin. Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography.

To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)), an endogenous micro-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D (1)H-NMR measurement and molecular modelling calculation. Both peptides were in equilibrium between the cis and trans rotamers around the Tyr--Pro w bond with population ratios of 1 : 1 to 1 : 2 in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans/cis rotamer ratio of 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure. Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans/cis rotamer form. On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH. Most of these folded conformers took an F1-type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH(3) (+)...COO(-)(Phe4), observed in its crystal structure. These results show that the substitution of a carboxyl group for the C-terminal amide group makes the peptide structure more flexible and leads to the ensemble of folded and open conformers. The conformational requirement of EM2 for binding to the micro-opioid receptor and the structural function of the C-terminal amide group are discussed on the basis of the present conformational features of EM2 and EM2OH and a possible model for binding to the micro-opioid receptor, constructed from the template structure of rhodopsin.

Amides↗
PubMed1996

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase.

The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angel restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time 1H-13C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally 13C-labelled sample was exploited for the stereospecific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C alpha, C', O) among the 25 conformers is 0.42 A for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel beta-strands arranged to form two perpendicular beta-sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core beta-strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764-769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short beta-strands in the regions Ser15 to Glu17 and Gly25 to Glu27 flanking the so-called "RT-Src' loop. This loop (residues Glu17 to Leu24), together with the "n-Src' loop (residues Gln37 to Ser46) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His14, Tyr16, Trp41, Phe54 and Phe59). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear 15N, 1H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.

Amino Acids↗
PubMed1994

Potential-energy calculations of terminally blocked tetrapeptides from the third loop of short-chain snake venom neurotoxins.

The conformational space of six tetrapeptides from the beta-bends in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations. It was shown that these peptides can be divided into two groups: those with a specific preference for an alpha-helix and those that exist as an ensemble of beta-turn conformations.

Amino Acid Sequence↗
PubMed2005

Structural and energetic origins of sequence-specific DNA bending: Monte Carlo simulations of papillomavirus E2-DNA binding sites.

DNA bending is an important structural feature for indirect readout in protein-DNA recognition. The binding of papillomavirus E2 transcription factors to their DNA binding sites is associated with DNA bending, providing an attractive model system to study the origins of sequence-specific DNA bending. The consensus E2 target is of the general form ACCGN(4)CGGT with a variable four base pair region. We applied a new all-atom Monte Carlo (MC) algorithm that combines effective sampling with fast conformational equilibration. The resulting MC ensembles resemble the corresponding high-resolution crystal structures very well. Distinct bending is observed for the E2-DNA binding site with a central AATT linker in contrast to an essentially straight DNA with a central ACGT linker. Contributions of specific base pair steps to the overall bending are shown in terms of local structural parameters. The analysis of conformational substates provides new insights into the energetic origins of intrinsic DNA bending.

Algorithms↗