Gap junction dynamics: reversible effects of hydrogen ions

Reversible crystallization of intramembrane particle packings is induced in gap junctions isolated from calf lens fibers by exposure to 3 x 10(-7) M or higher [H+] (pH 6.5 or lower). The changes from disordered to crystalline particle packings induced by low pH are similar to those produced in junctions of intact cells by uncoupling treatments, indicating that H+, like divalent cations, could be an uncoupling agent. The freeze-fracture appearance of both control and low pH-treated gap junctions is not altered by glutaraldehyde fixation and cryoprotective treatment, as suggested by experiments in which gap junctions of both intact cells and isolated fractions are freeze- fractured after rapid freezing to liquid N2 temperature according to Heuser et al. (13). In junctions exposed to low pH, the particles most often form orthogonal and rhombic arrays, frequently fused with each other. A number of structural characteristics of these arrays suggest that the particles of lens fiber gap junctions may be shaped as tetrameres.     

Molecular dynamics simulations of synthetic peptide folding

Because the time scale of protein folding is much greater than that of the widely used simulations of native structures, a detailed report of molecular dynamics simulations of folding has not been available. In this study, we Included the average solvent effect in the potential functions to simplify the calculation of the solvent effect and carried out long molecular dynamics simulations of the alanine-based synthetic peptides at 274 K. From either an extended or a randomly generated conformation, the simulations approached a helix-coil equilibrium in about 3 ns. The multiple minima problem did not prevent helix folding. The calculated helical ratio of Ac-AAQAAAAQAAAAQAAY-NH₂ was 47%, in good agreement with the circular dichroism measurement (about 50%). A helical segment with frayed ends was the most stable conformation, but the hydrophobic interaction favored the compact, distorted helix-turn-helix conformations. The transition between the two types of conformations occurred in a much larger time scale than helix propagation. The transient hydrogen bonds between the glutamine side chain and the backbone carbonyl group could reduce the free energy barrier of helix folding and unfolding. The substitution of a single alanine residue in the middle of the peptide with valine or glycine decreased the average helical ratio significantly, in agreement with experimental observations. © 1996 Wiley-Liss, Inc.     

Characterization of peptide folding nuclei by hydrogen/deuterium exchange-mass spectrometry

Covalently linked pairs of well-chosen peptides can be good model systems for protein folding studies because they can adopt stable secondary, side-chain, and tertiary structure under certain conditions. We demonstrate a method for characterizing the structure in such peptide pairs by hydrogen/deuterium exchange of individual amide groups analyzed by collision-induced dissociation tandem mass spectrometry, in concert with circular dichroism spectroscopy. We apply the method to two peptides (and their three possible pairs) from bovine pancreatic trypsin inhibitor to address specific hypotheses regarding the stabilization of local secondary structure by long-range interactions.     

A directed essential dynamics simulation of peptide folding

We present a directed essential dynamics (DED) method for peptide and protein folding. DED is a molecular dynamics method based on the essential dynamics sampling and the principal component analysis. The main idea of DED is to use principal component analysis to determine the direction of the most active collective motion of peptides at short intervals of time (20 fs) during the folding process and then add an additional force along it to adjust the folding direction. This method can make the peptides avoid being trapped in the local minima for a long time and enhance the sampling efficiency in conformational space during the simulation. An S-peptide with 15 amino acids is used to demonstrate the DED method. The results show that DED can lead the S-peptide to fold quickly into the native state, whereas traditional molecular dynamics needs more time to do this.     

Photoinduced conformational dynamics of a photoswitchable peptide: a nonequilibrium molecular dynamics simulation study

Employing nonequilibrium molecular dynamics simulations, a comprehensive computational study of the photoinduced conformational dynamics of a photoswitchable bicyclic azobenzene octapeptide is presented. The calculation of time-dependent probability distributions along various global and local reaction coordinates reveals that the conformational rearrangement of the peptide is rather complex and occurs on at least four timescales: 1) After photoexcitation, the azobenzene unit of the molecule undergoes nonadiabatic photoisomerization within 0.2 ps. 2) On the picosecond timescale, the cooling (13 ps) and the stretching (14 ps) of the photoexcited peptide is observed. 3) Most reaction coordinates exhibit a 50-100 ps component reflecting a fast conformational rearrangement. 4) The 500-1000 ps component observed in the simulation accounts for the slow diffusion-controlled conformational equilibration of the system. The simulation of the photoinduced molecular processes is in remarkable agreement with time-resolved optical and infrared experiments, although the calculated cooling as well as the initial conformational rearrangements of the peptide appear to be somewhat too slow. Based on an ab initio parameterized vibrational Hamiltonian, the time-dependent amide I frequency shift is calculated. Both intramolecular and solvent-induced contributions to the frequency shift were found to change by < or = 2 cm(-1), in reasonable agreement with experiment. The potential of transient infrared spectra to characterize the conformational dynamics of peptides is discussed in some detail.     

Desolvation shell of hydrogen bonds in folded proteins,protein complexes and folding pathways

A few backbone hydrogen bonds (HBS) in native protein folds are poorly protected from water attack: their desolvation shell contains an inordinately low number of hydrophobic residues. Thus, an approach by solvent-structuring moieties of a binding partner should contribute significantly to enhance their stability. This effect represents an important factor in the site specificity inherent to protein binding, as inferred from a strong correlation between poorly desolvated HBs and binding sites. The desolvation shells were also examined in a dynamic context: except for a few singular under-protected bonds, the size of desolvation shells is preserved along the folding trajectory.     

The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.     

Structured pathway across the transition state for peptide folding revealed by molecular dynamics simulations

Small globular proteins and peptides commonly exhibit two-state folding kinetics in which the rate limiting step of folding is the surmounting of a single free energy barrier at the transition state (TS) separating the folded and the unfolded states. An intriguing question is whether the polypeptide chain reaches, and leaves, the TS by completely random fluctuations, or whether there is a directed, stepwise process. Here, the folding TS of a 15-residue β-hairpin peptide, Peptide 1, is characterized using independent 2.5 μs-long unbiased atomistic molecular dynamics (MD) simulations (a total of 15 μs). The trajectories were started from fully unfolded structures. Multiple (spontaneous) folding events to the NMR-derived conformation are observed, allowing both structural and dynamical characterization of the folding TS. A common loop-like topology is observed in all the TS structures with native end-to-end and turn contacts, while the central segments of the strands are not in contact. Non-native sidechain contacts are present in the TS between the only tryptophan (W11) and the turn region (P7-G9). Prior to the TS the turn is found to be already locked by the W11 sidechain, while the ends are apart. Once the ends have also come into contact, the TS is reached. Finally, along the reactive folding paths the cooperative loss of the W11 non-native contacts and the formation of the central inter-strand native contacts lead to the peptide rapidly proceeding from the TS to the native state. The present results indicate a directed stepwise process to folding the peptide.     

Synthesis of gold structures by gold-binding peptide governed by concentration of gold ion and peptide

Although biological synthesis methods for the production of gold structures by microorganisms, plant extracts, proteins, and peptide have recently been introduced, there have been few reports pertaining to controlling their size and morphology. The gold ion and peptide concentrations affected on the size and uniformity of gold plates by a gold-binding peptide Midas-11. The higher concentration of gold ions produced a larger size of gold structures reached 125.5 μm, but an increased amount of Midas-11 produced a smaller size of gold platelets and increased the yield percentage of polygonal gold particles rather than platelets. The mechanisms governing factors controlling the production of gold structures were primarily related to nucleation and growth. These results indicate that the synthesis of gold architectures can be controlled by newly isolated and substituted peptides under different reaction conditions.     

Structure and hydrogen bonds of cyclohexapeptide RA-VII by molecular dynamics simulations and quantum chemical calculations

We computationally examined the structure of anti-tumour bicyclic hexapeptide RA-VII. This peptide adopts three conformations (confs.), A, B and C, in dimethyl sulfoxide (DMSO). Although it was experimentally reported that the structure of conf. A is important for anti-tumour activity, the dynamics of confs. A, B and C are not well known. We performed quantum chemical calculations and molecular dynamics (MD) simulations of RA-VII in DMSO. The MD simulations indicated two different local stable structures for conf. C: a structure containing a bent 18-membered ring and another structure containing a rotated peptide bond between Tyr⁶ and d-Ala¹. The root-mean-square fluctuation of the 14-membered ring for conf. A was larger than that for confs. B and C. Ala⁴ formed intramolecular hydrogen bonds more often in conf. A than in the other conformations. A large number of hydrogen bonds and large structural fluctuations are important for the anti-tumour activity of RA-VII. Our results for the structural change of conf. C and the analysis of the dynamics for confs. A, B and C may contribute to the design of new analogues of cyclic peptides.     

Molecular dynamics simulation of protein folding by essential dynamics sampling: folding landscape of horse heart cytochrome c

A new method for simulating the folding process of a protein is reported. The method is based on the essential dynamics sampling technique. In essential dynamics sampling, a usual molecular dynamics simulation is performed, but only those steps, not increasing the distance from a target structure, are accepted. The distance is calculated in a configurational subspace defined by a set of generalized coordinates obtained by an essential dynamics analysis of an equilibrated trajectory. The method was applied to the folding process of horse heart cytochrome c, a protein with approximately 3000 degrees of freedom. Starting from structures, with a root-mean-square deviation of approximately 20 A from the crystal structure, the correct folding was obtained, by utilizing only 106 generalized degrees of freedom, chosen among those accounting for the backbone carbon atoms motions, hence not containing any information on the side chains. The folding pathways found are in agreement with experimental data on the same molecule.     

Cooperative folding mechanism of a beta-hairpin peptide studied by a multicanonical replica-exchange molecular dynamics simulation

G-peptide is a 16-residue peptide of the C-terminal end of streptococcal protein G B1 domain, which is known to fold into a specific beta-hairpin within 6 micros. Here, we study molecular mechanism on the stability and folding of G-peptide by performing a multicanonical replica-exchange (MUCAREM) molecular dynamics simulation with explicit solvent. Unlike the preceding simulations of the same peptide, the simulation was started from an unfolded conformation without any experimental information on the native conformation. In the 278-ns trajectory, we observed three independent folding events. Thus MUCAREM can be estimated to accelerate the folding reaction more than 60 times than the conventional molecular dynamics simulations. The free-energy landscape of the peptide at room temperature shows that there are three essential subevents in the folding pathway to construct the native-like beta-hairpin conformation: (i) a hydrophobic collapse of the peptide occurs with the side-chain contacts between Tyr45 and Phe52, (ii) then, the native-like turn is formed accompanying with the hydrogen-bonded network around the turn region, and (iii) finally, the rest of the backbone hydrogen bonds are formed. A number of stable native hydrogen bonds are formed cooperatively during the second stage, suggesting the importance of the formation of the specific turn structure. This is also supported by the accumulation of the nonnative conformations only with the hydrophobic cluster around Tyr45 and Phe52. These simulation results are consistent with high phi-values of the turn region observed by experiment.     

Cell-permeable chameleonic peptides: Exploiting conformational dynamics in de novo cyclic peptide design

Membrane-traversing peptides offer opportunities for targeting intracellular proteins and oral delivery. Despite progress in understanding the mechanisms underlying membrane traversal in natural cell-permeable peptides, there are still several challenges to designing membrane-traversing peptides with diverse shapes and sizes. Conformational flexibility appears to be a key determinant of membrane permeability of large macrocycles. We review recent developments in the design and validation of chameleonic cyclic peptides, which can switch between alternative conformations to enable improved permeability through cell membranes, while still maintaining reasonable solubility and exposed polar functional groups for target protein binding. Finally, we discuss the principles, strategies, and practical considerations for rational design, discovery, and validation of permeable chameleonic peptides.