Molecular dynamics of oligopeptides. 3. Maps of levels of free energy of modified dipeptides and dynamic correlation in amino acid residues]

A method of free energy maps for studying the dynamic correlations of fluctuations in molecules with conformational mobility is proposed. An agreement between the structure of the free energy level map and the type of the corresponding cross-correlation function (in the presence and absence of the correlation of fluctuations in conformational freedom degree in modified dipeptides) was established.     

Photomodulation of conformational states. Synthesis of cyclic peptides with backbone-azobenzene moieties.

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4-aminomethyl)phenylazobenzoic acid (H-AMPB-OH) and (4-amino)phenylazobenzoic acid (H-APB-OH) were synthesized and used to cyclize a bis-cysteinyl-octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H-AMPB-OH proceeded smoothly on a chlorotrityl-resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H-APB-OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S-tert-butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S-trityl protection. Side-chain cyclization of the APB-peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side-chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly.     

Comparison of the Conformational Behavior of Sialyllactose Complexed with the two Viral Attachment Proteins Influenza A Hemagglutinin and the Murine Polyomavirus

The attachment of a virus to the host cell surface is influenced by enthalpic and entropic factors. A detailed evaluation of all possible energetic interactions including the effects of solvent molecules seems to be a promising way to gain deeper insights in the overall process of binding. Here we performed intensive molecular dynamics studies to compare the conformational space available for the unbound sialyllactose in aqueous solution and when complexed with influenza A hemagglutinin and the murine polyoma virus. In general the conformational freedom of sialyllactose is considerably reduced compared to the free state. Remarkably, two different conformations of the Siaα(2-3)Galβ glycosidic linkages are preferred (which are both populated in the free state) when complexed with either protein.     

A syn conformation for inosine, the wobble nucleoside in some tRNA's

The crystal structure of an orthorhombic form of inosine was determined from three-dimensional X-ray diffraction data. There are two crystallographically independent inosine molecules, both of which assume a $\text{syn}$ conformation that is different from the conformations found in other crystalline forms of inosine. Apparently, inosine has considerable conformational freedom, a property that may be required at the “wobble” position of anticodon triplets.     

Computational study of the conformational profiles of model bis-cystine cyclic peptides

Bis-cystine cyclic peptides are a new kind of molecules with potential use as cavitands, transporters or antagonists of target ligands. Studies aimed at establishing their conformational profiles may prove useful in understanding their characteristics and potentiate their use in molecular design. The present investigation reports the results of a computational study devoted to establishing the conformational preferences of model bis-cystine cyclic peptides and the properties in common with their linear analogs. For this purpose a study of four model compounds: (Ac-Cys-X-Cys-NHMe)₂ and (Ac-Cys-X-X-Cys-NHMe)₂ with X = Ala, Val, was performed. The goal of the study was to explore the importance of the conformational nature of the central residues, the effect of the number of them, and the loss of conformational freedom after cyclization on model molecules. Accordingly, the conformational space and the dynamic behaviour of the four cyclic peptides as well as the corresponding linear analogs was carefully explored. The results indicate the existence of structural patterns that might be useful for the use of this kind of molecule in de novo molecular design     

A novel four-amino acid determinant defines conformational freedom within chorionic gonadotropin beta-subunits

On the basis of apparent molecular mass heterogeneity following reducing versus nonreducing SDS-PAGE, we determined that the beta-subunit of macaque (Macaca fascicularis) chorionic gonadotropin (mCG-beta) is more conformationally constrained than the beta-subunit of human chorionic gonadotropin (hCG-beta). The amino acid sequences of these two subunits are 81% identical. To determine the conformational variance source, which was not due to glycosylation differences, we generated a series of hCG-beta-mCG-beta chimeras and identified domains that contributed to CG-beta conformational freedom. We discovered that the CG-beta 54-101 domain contained a small subdomain, residues 74-77, that regulated the conformational freedom of the beta-subunit; i.e., when residues 74-77 were of macaque origin (PGVD), the mutated hCG-beta subunit displayed macaque-like conformational rigidity, and when residues 74-77 were of human origin (RGVN), the mutated mCG-beta subunit displayed human-like conformational freedom and microheterogeneity. Additionally, CG-beta N-terminal domain residues (8, 18, 42, and 46-48) were also found to influence CG-beta conformational freedom when residues 74-77 were of human but not macaque origin. The biological significance of the CG-beta conformational variance was tested using a biological assay that showed that the hCG-alpha-hCG-beta heterodimer facilitated human CG receptor-mediated cAMP-driven luciferase reporter gene activity in HEK cells nearly 1 order of magnitude more effectively than the hCG-alpha-mCG-beta chimera. Together, these data demonstrate that two essential amino acid residues within a four-amino acid subdomain regulated CG-beta conformational freedom and that a conformational difference between hCG-beta and mCG-beta was recapitulated in the context of receptor-mediated CG heterodimer signal transduction activation.     

Structural Plasticity of Neurexin 1α: Implications for its Role as Synaptic Organizer

α-Neurexins are synaptic organizing molecules implicated in neuropsychiatric disorders. They bind and arrange an array of different partners in the synaptic cleft. The extracellular region of neurexin 1α (n1α) contains six LNS domains (L1–L6) interspersed by three Egf-like repeats. N1α must encode highly evolved structure–function relationships in order to fit into the narrow confines of the synaptic cleft, and also recruit its large, membrane-bound partners. Internal molecular flexibility could provide a solution; however, it is challenging to delineate because currently no structural methods permit high-resolution structure determination of large, flexible, multi-domain protein molecules. To investigate the structural plasticity of n1α, in particular the conformation of domains that carry validated binding sites for different protein partners, we used a panel of structural techniques. Individual particle electron tomography revealed that the N-terminally and C-terminally tethered domains, L1 and L6, have a surprisingly limited range of conformational freedom with respect to the linear central core containing L2 through L5. A 2.8-Å crystal structure revealed an unexpected arrangement of the L2 and L3 domains. Small-angle X-ray scattering and electron tomography indicated that incorporation of the alternative splice insert SS6 relieves the restricted conformational freedom between L5 and L6, suggesting that SS6 may work as a molecular toggle. The architecture of n1α thus encodes a combination of rigid and flexibly tethered domains that are uniquely poised to work together to promote its organizing function in the synaptic cleft, and may permit allosterically regulated and/or concerted protein partner binding.     

Enhanced sampling of molecular dynamics simulation of peptides and proteins by double coupling to thermal bath

Low sampling efficiency in conformational space is the well-known problem for conventional molecular dynamics. It greatly increases the difficulty for molecules to find the transition path to native state, and costs amount of CPU time. To accelerate the sampling, in this paper, we re-couple the critical degrees of freedom in the molecule to environment temperature, like dihedrals in generalized coordinates or nonhydrogen atoms in Cartesian coordinate. After applying to ALA dipeptide model, we find that this modified molecular dynamics greatly enhances the sampling behavior in the conformational space and provides more information about the state-to-state transition, while conventional molecular dynamics fails to do so. Moreover, from the results of 16 independent 100?ns simulations by the new method, it shows that trpzip2 has one-half chances to reach the naive state in all the trajectories, which is greatly higher than conventional molecular dynamics. Such an improvement would provide a potential way for searching the conformational space or predicting the most stable states of peptides and proteins.     

Biological complexity,quantum coherent states and the problem of efficient transmission of information inside a cell

The intracellular channel of information transmission was analyzed from the point of view of complexity. The most important steps in the transfer of information within a cell are the folding, transport and recognition of proteins. It was shown that the large number of conformational degrees of freedom that proteins possess can paradoxically lead to an information channel with an exponentially small capacity. To resolve this paradox, a model, which assumes a quantum collective behavior of biologically important molecules, was proposed. Experiments to test the quantum nature of the intracellular transfer of information were also proposed.     

Ribonuclease A suggests how proteins self-chaperone against amyloid fiber formation

Genomic analyses have identified segments with high fiber-forming propensity in many proteins not known to form amyloid. Proteins are often protected from entering the amyloid state by molecular chaperones that permit them to fold in isolation from identical molecules; but, how do proteins self-chaperone their folding in the absence of chaperones? Here, we explore this question with the stable protein ribonuclease A (RNase A). We previously identified fiber-forming segments of amyloid-related proteins and demonstrated that insertion of these segments into the C-terminal hinge loop of nonfiber-forming RNase A can convert RNase A into the amyloid state through three-dimensional domain-swapping, where the inserted fiber-forming segments interact to create a steric zipper spine. In this study, we convert RNase A into amyloid-like fibers by increasing the loop length and hence conformational freedom of an endogenous fiber-forming segment, SSTSAASS, in the N-terminal hinge loop. This is accomplished by sandwiching SSTSAASS between inserted Gly residues. With these inserts, SSTSAASS is now able to form the steric zipper spine, allowing RNase A to form amyloid-like fibers. We show that these fibers contain RNase A molecules retaining their enzymatic activity and therefore native-like structure. Thus, RNase A appears to prevent fiber formation by limiting the conformational freedom of this fiber-forming segment from entering a steric zipper. Our observations suggest that proteins have evolved to self-chaperone by using similar protective mechanisms.     

Mass-weighted molecular dynamics simulation of cyclic polypeptides.

A modified molecular dynamics (MD) method in which atomic masses are weighted was developed previously for studying the conformational flexibility of neuroregulating tetrapeptide Phe-Met-Arg-Phe-amide (FMRF-amide). The method has now been applied to longer and constrained molecules, namely a disulfide-linked cyclic hexapeptide, c[CYFQNC], and its linear and "pseudo-cyclic" analogues. The sampling of dehedral conformational space of teh linear hexapeptide in mass-weighted MD simulations was found to be improved significantly over conventional MD simulations, as in the case of the shorter FMRF-amide molecule studied previously. In the cyclic hexapeptide, the internal constraint of the molecule due to the intramolecular disulfide bond (hence the absence of free terminals in the molecule) does not adversely affect the significant improvement of conformational sampling in mass-weighted MD simulations over normal MD simulations. The pseudo-cyclic polypeptide is identical to the linear CYFQNC molecule in amino acid sequence (i.e., side chains of the cysteine residues are reduced), but the positions of its two terminal heavy atoms were held fixed in space such that the molecule has a nearly cyclic conformation. For this molecule, the mass-weighted MD simulation generated a wide range of polypeptide backbone conformations covering the internal dihedral degrees of freedom; moreover, the physical space of the pseudo-cyclic structure was also sampled in a complete revolution of the entire molecular fragment about the two fixed termini during the simulation. These characteristics suggest that mass-weighted MD can also be an extremely useful method for conformational analyses of constrained molecules and, in particular, for modeling loops on protein surfaces.     

Crystal structure and solid state 13C NMR analysis of nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-gluco- and D-galactopyranosides

The X-ray diffraction analysis of o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside (1), m-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, p-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside was performed. It was found that except in the case of 1, all other crystals have one molecule in the independent part of the crystal unit cell. The results support the opinion that the nitro group does not conjugate effectively with the phenyl ring. In the 13C CP MAS spectrum of 1 the signals are split, confirming the presence of two independent molecules. Similarly, the 13C CP MAS NMR spectrum of p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside indicated the presence of two non-equivalent molecules in the crystal unit. One of these molecules has more conformational freedom enabling rotation of the phenyl ring.     

Spatial organization of the N-terminal beta-endorphin tridecapeptide

Using conformational analysis spatial structure and conformational properties of the N-terminal tridecapeptide--endorphine molecules were investigated. Calculations were based on the fragmental analysis using non-valent, electrostatic, torsional interactions and hydrogen bonds. It was shown that tridecapeptide could exist in several low-energetical conformational states. Enkephaline fragments structure depends on the most perspective structure of free metioninenkephaline. The results can be used for conformational analysis of endorphine molecules, for structure--function relations study.     

Molecular dynamics simulations of forced conformational transitions in 1,6-linked polysaccharides

Recent atomic force microscopy stretching measurements of single polysaccharide molecules suggest that their elasticity is governed by force-induced conformational transitions of the pyranose ring. However, the mechanism of these transitions and the mechanics of the pyranose ring are not fully understood. Here we use steered molecular dynamics simulations of the stretching process to unravel the mechanism of forced conformational transitions in 1,6 linked polysaccharides. In contrast to most sugars, 1,6 linked polysaccharides have an extra bond in their inter-residue linkage, C5-C6, around which restricted rotations occur and this additional degree of freedom increases the mechanical complexity of these polymers. By comparing the computational results with the atomic force microscopy data we determine that forced rotations around the C5-C6 bond have a significant and different impact on the elasticity of alpha- and beta-linked polysaccharides. Beta-linkages of a polysaccharide pustulan force the rotation around the C5-C6 bonds and produce a Hookean-like elasticity but do not affect the conformation of the pyranose rings. However, alpha-linkages of dextran induce compound conformational transitions that include simultaneous rotations around the C5-C6 bonds and chair-boat transitions of the pyranose rings. These previously not-recognized transitions are responsible for the characteristic plateau in the force-extension relationship of dextran.     

Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity

Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein–ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i) prediction of enzyme specificity altering mutations and (ii) prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art “fixed backbone” design methods perform poorly on these tests, we develop a new “coupled moves” design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein – ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution.     

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.     

Cold unfolding of β-hairpins: a molecular-level rationalization

Isolated β-hairpins in water have a temperature dependence of their conformational stability qualitatively resembling that of globular proteins, showing both cold and hot unfolding transitions. It is shown that a molecular-level rationalization of this cold unfolding can be provided extending the approach devised for globular proteins (Graziano G. Phys Chem Chem Phys 2010; 12:14245-14252). The decrease in the solvent-excluded volume upon folding, measured by the decrease in the solvent accessible surface area, produces a gain in configurational/translational entropy of water molecules that is the main stabilizing contribution of the folded conformation. This always stabilizing Gibbs energy contribution has a parabolic-like temperature dependence in water and is exactly counterbalanced at two temperatures (i.e., the cold and hot unfolding temperatures) by the always destabilizing Gibbs energy contribution due to the loss in conformational degrees of freedom of the peptide chain.     

Energy minimization in low-frequency normal modes to efficiently allow for global flexibility during systematic protein-protein docking

Protein-protein association can frequently involve significant backbone conformational changes of the protein partners. A computationally rapid method has been developed that allows to approximately account for global conformational changes during systematic protein-protein docking starting from many thousands of start configurations. The approach employs precalculated collective degrees of freedom as additional variables during protein-protein docking minimization. The global collective degrees of freedom are obtained from normal mode analysis using a Gaussian network model for the protein. Systematic docking searches were performed on 10 test systems that differed in the degree of conformational change associated with complex formation and in the degree of overlap between observed conformational changes and precalculated flexible degrees of freedom. The results indicate that in case of docking searches that minimize the influence of local side chain conformational changes inclusion of global flexibility can significantly improve the agreement of the near-native docking solutions with the corresponding experimental structures. For docking of unbound protein partners in several cases an improved ranking of near native docking solutions was observed. This was achieved at a very modest ( approximately 2-fold) increase of computational demands compared to rigid docking. For several test cases the number of docking solutions close to experiment was also significantly enhanced upon inclusion of soft collective degrees of freedom. This result indicates that inclusion of global flexibility can facilitate in silico protein-protein association such that a greater number of different start configurations results in favorable complex formation.