Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein α-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R_H) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R_H value was shifted to 100, indicating the aggregation of denatured xylanase. The α-crystallin·xylanase complex exhibited the recovery of functional activity with the extent of ~43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.     

Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

A 10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are located in a stable helical region of a transmembrane peptide. The 25-residue peptide (sMTM7) used mimics the cytoplasmic proton hemichannel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The three-dimensional structure of peptide sMTM7 in DMSO has been previously solved by NMR spectroscopy. The radial and spatial distributions of the DMSO molecules surrounding the peptide as well as the number of hydrogen bonds between DMSO and the side chains of the amino acid residues involved are extracted from the molecular dynamics simulations. Analysis of the molecular dynamics trajectories shows that the amino acid side chains are fully embedded in DMSO. Polar and positively charged amino acid side chains have dipole-dipole interactions with the oxygen atom of DMSO and form hydrogen bonds. Apolar residues become solvated by DMSO through the formation of a hydrophobic pocket in which the methyl groups of DMSO are pointing toward the hydrophobic side chains of the residues involved. The dual solvation properties of DMSO cause it to be a good membrane-mimicking solvent for transmembrane peptides that do not unfold due to the presence of DMSO.     

Solubilization of Paclitaxel (taxol) by peptoad self-assemblies

A molecular dynamics simulation was carried out involving a paclitaxel molecule, 987 peptoad molecules, and 35 938 water molecules (conditions shown experimentally to effect paclitaxel solubilization in water). The peptoads are shown to form large clumps, the centers of which are dry and thus favorable to hydrogen bonding between paclitaxel and peptoads. Hydrogen-bonding equilibrium among the peptoads themselves in the developing clumps is achieved in 2 ns. The number and position of hydrogen bonds between the paclitaxel and peptoads fluctuate randomly from two to six within a 2-5 ns time frame. Hydrophobic association between the peptoad chains and the apolar paclitaxel groups does not seem to be an important element of the solubilization. Instead, the hydrophobic chains of the peptoads encasing the paclitaxel extend outward into the dry interior of the peptoad clump where other chains in the clump are located. One hopes that studies such as this will ultimately allow rational predictions when designing new and specific drug solubilizers.     

Monolayer formation of short helical turn forming peptide derivatives at the air–water and air–solid interfaces

Two tetrapeptide derivatives [peptide A (Boc–Ala–Ile–Ile–Gly–OMe) and peptide B (Boc–Ala–Ile–Leu–Ser–OMe)], that take helical turn conformation in solution, were shown to form monolayer at the air/water interface. Circular dichroism (CD) measurements indicate that peptide A has more helical turn propensity than peptide B in sodium dodecyl sulphate (SDS) micelles. Langmuir–Blodgget film study of peptides A and B suggest that both the peptides form stable monolayer at the air/water interface. Spectroscopic investigations reveal that peptide A forms helical turn assemblage on transferring the film into hydrophilic quartz and hydrophobic ZnSe surfaces. Whereas, peptide B adopts β-sheet structure on hydrophilic surface and a mixture of β-sheet and helical turn conformation on hydrophobic surface.     

Concentration enrichment of urea at cellulose surfaces: results from molecular dynamics simulations and NMR spectroscopy

A combined solid-state NMR and Molecular Dynamics simulation study of cellulose in urea aqueous solution and in pure water was conducted. It was found that the local concentration of urea is significantly enhanced at the cellulose/solution interface. There, urea molecules interact directly with the cellulose through both hydrogen bonds and favorable dispersion interactions, which seem to be the driving force behind the aggregation. The CP/MAS ¹³C spectra was affected by the presence of urea at high concentrations, most notably the signal at 83.4 ppm, which has previously been assigned to C4 atoms in cellulose chains located at surfaces parallel to the (110) crystallographic plane of the cellulose Iβ crystal. Also dynamic properties of the cellulose surfaces, probed by spin-lattice relaxation time ¹³CT ₁ measurements of C4 atoms, are affected by the addition of urea. Molecular Dynamics simulations reproduce the trends of the T ₁ measurements and lends new support to the assignment of signals from individual surfaces. That urea in solution is interacting directly with cellulose may have implications on our understanding of the mechanisms behind cellulose dissolution in alkali/urea aqueous solutions.     

Responses of Transmembrane Peptide and Lipid Chains to Hydrophobic Mismatch

Hydrophobic mismatch between the hydrophobic length of membrane proteins and hydrophobic thickness of membranes is a crucial factor in controlling protein function and assembly. We combined fluorescence with circular dichroism(CD) and attenuated total reflection infrared(ATR-IR) spectroscopic methods to investigate the behaviors of the peptide and lipids under hydrophobic mismatch using a model peptide from the fourth transmembrane domain of natural resistance-associated macrophage protein 1(Nramp1), the phosphatidylcholines(PCs) and phosphatidylglycerols(PGs) with different lengths of acyl chains(14:0, 16:0 and 18:0). In all PG lipid membranes, the peptide forms stable a-helix structure, and the helix axis is parallel to lipid chains. The helical span and orientation hardly change in varying thickness of PG membranes, while the lipid chains can deform to accommodate to the hydrophobic surface of embedded peptide. By comparison, the helical structures of the model peptide in PC lipid membranes are less stable. Upon incorporation with PC lipid membranes, the peptide can deform itself to accommodate to the hydrophobic thickness of lipid membranes in response to hydrophobic mismatch. In addition, hydrophobic mismatch can increase the aggregation propensity of the peptide in both PC and PG lipid membranes and the peptide in PC membranes has more aggregation tendency than that in PG membranes.     

Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations

Lipid-modified membrane-binding proteins are essential in signal transduction events of the cell, a typical example being the GTPase ras. Recently, membrane binding of a doubly lipid-modified heptapeptide from the C-terminus of the human N-ras protein was studied by spectroscopic techniques. It was found that membrane binding is mainly due to lipid chain insertion, but it is also favored by interactions between apolar side chains and the hydrophobic region of the membrane. Here, 10 explicit solvent molecular dynamics simulations for a total time of about 150 ns are used to investigate the atomic details of the peptide-membrane association. The 16:0 peptide lipid chains are more mobile than the 14:0 phospholipid chains, which is in agreement with (2)H NMR experiments. Peptide-lipid and peptide-solvent interactions, backbone and side-chain distributions, as well as the effects of lipidated peptide insertion onto the structure, and dynamics of a 1,2-dimyristoylglycero-3-phosphocholine bilayer are described. The simulation results validate the structural model proposed by the analysis of spectroscopic data and highlight the main aspects of the insertion mechanism. The peptide in the membrane is rather rigid over the simulation time scale of about 10 ns, but different partially extended conformations devoid of backbone hydrogen bonds are observed in different trajectories.     

Tunable helical assemblies of L-alanine methyl ester-containing polyphenylacetylene

The self-assemblying behaviors of L-alanine methyl ester-containing polyphenylacetylene (PPA-Ala, in Chart 1 ) were investigated upon the evaporation of its solvent on mica and on air/water interfaces. The introduction of chiral amino acid attachments to the polyphenylacetylene backbone induced a helical conformation of the backbone, which was stabilized by various noncovalent interactions, especially hydrophobic effect and hydrogen bonds. The helicity of the polymer was further amplified in its higher-order self-assemblies as the formation of helical fibers on the surface of mica upon natural evaporation of its THF solution. By LB technique, the polymer chains were guided to form ordered parallel ridges and highly aligned, with their helical conformation still remaining. The reorganization of the chiral polymer chains on air/water interface was associated with the additional hydrophobic effect of PPA-Ala on an air/water interface. The polymer backbones had to adopt different arrangements to minimize their contact with water, and this adjustment led to the formation of aligned polymer ridges under proper surface pressure.     

Organic Ligand Binding by a Hydrophobic Cavity in a Designed Tetrameric Coiled‐Coil Protein

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled‐coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand‐binding characteristics of the cavities, we originally designed two other coiled‐coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size‐complementary organic compounds. The dissociation constants, K_d, of AM2W were 220 nM for adamantane, 81 μM for 1‐adamantanol, and 294 μM for 1‐adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.     

Effects of Curcumin and Ferulic Acid on the Folding of Amyloid-β Peptide

The polyphenols curcumin (CU) and ferulic acid (FA) are able to inhibit the aggregation of amyloid-β (Aβ) peptide with different strengths. CU is a strong inhibitor while FA is a weaker one. In the present study, we examine the effects of CU and FA on the folding process of an Aβ monomer by 1 µs molecular dynamics (MD) simulations. We found that both inhibitors increase the helical propensity and decrease the non-helical propensity of Aβ peptide. They prevent the formation of a dense bulk core and shorten the average lifetime of intramolecular hydrogen bonds in Aβ. CU makes more and longer-lived hydrogen bonds, hydrophobic, π–π, and cation–π interactions with Aβ peptide than FA does, which is in a good agreement with the observed stronger inhibitory activity of CU on Aβ aggregation.     

Aqueous urea solutions: structure, energetics, and urea aggregation

Urea is ubiquitously used as a protein denaturant. To study the structure and energetics of aqueous urea solutions, we have carried out molecular dynamics simulations for a wide range of urea concentrations and temperatures. The hydrogen bonds between urea and water were found to be significantly weaker than those between water molecules, which drives urea self-aggregation due to the hydrophobic effect. From the reduction of the water exposed urea surface area, urea was found to exhibit an aggregation degree of ca. 20% at concentrations commonly used for protein denaturation. Structurally, three distinct urea pair conformations were identified and their populations were analyzed by translational and orientational pair distribution functions. Furthermore, urea was found to strengthen water structure in terms of hydrogen bond energies and population of solvation shells. Our findings are consistent with a direct interaction between urea and the protein as the main driving force for protein denaturation. As an additional, more indirect effect, urea was found to enhance water structure, which would suggest a weakening of the hydrophobic effect.     

Molecular dynamics simulations of pro-apoptotic BH3 peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein Bcl-X<Subscript>L</Subscript>

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the intrinsic pathway of apoptosis. Interactions between specific anti- and pro-apoptotic Bcl-2 proteins determine the fate of a cell. Anti-apoptotic Bcl-2 proteins have been shown to be over-expressed in certain cancers and they are attractive targets for developing anti-cancer drugs. Peptides from the BH3 region of pro-apoptotic proteins have been shown to interact with anti-apoptotic Bcl-2 proteins and induce biological activity similar to that observed in parent proteins. However, the specificity of BH3 peptides derived from different pro-apoptotic proteins differ for different anti-apoptotic Bcl-2 proteins. In this study, we have investigated the relationship between the stable helical nature of BH3 peptides and their affinities to Bcl-X_L, an anti-apoptotic Bcl-2 protein. We have carried out molecular dynamics simulations of six BH3 peptides derived from Bak, Bad and Bim pro-apoptotic proteins for a period of 50 ns each in aqueous medium. Due to the amphipathic nature of BH3 peptides, the hydrophobic residues on the hydrophobic face tend to cluster together in all BH3 peptides. While this process resulted in a complete loss of helical structure in 16-mer Bak and 16-mer Bad wild type peptides, stabilizing interactions in the hydrophilic face of the BH3 peptides and capping interactions helped to maintain partial helical character in 16-mer Bad mutant and 16-mer Bim peptides. The latter two 16-mer peptides exhibit higher affinity for Bcl-X_L. Similarly the longer BH3 peptides, 25-mer Bad and 33-mer Bim, also resulted in smaller and stable helical fragments and their helical conformation is stabilized by interactions between residues in the solvent-exposed hydrophilic half of the peptide. The stable nature of helical segment in a BH3 peptide can be directly correlated to its binding affinity and the helical region encompassed the highly conserved Leu residue. We propose that upon approaching the hydrophobic groove of anti-apoptotic proteins, a longer helix will be induced in high affinity BH3 peptides by extending the smaller stable helical segments around the conserved Leu residue in both N- and C-terminal regions. The results reported in this study will have implications in developing peptide-based inhibitors for anti-apoptotic Bcl-2 proteins.     

Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study.

The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.     

The effect of aqueous solutions of trimethylamine-N-oxide on pressure induced modifications of hydrophobic interactions

To understand the mechanism of protein protection by the osmolyte trimethylamine-N-oxide (TMAO) at high pressure, using molecular dynamics (MD) simulations, solvation of hydrophobic group is probed in aqueous solutions of TMAO over a wide range of pressures relevant to protein denaturation. The hydrophobic solute considered in this study is neopentane which is a considerably large molecule. The concentrations of TMAO range from 0 to 4 M and for each TMAO concentration, simulations are performed at five different pressures ranging from 1 atm to 8000 atm. Potentials of mean force are calculated and the relative stability of solvent-separated state over the associated state of hydrophobic solute are estimated. Results suggest that high pressure reduces association of hydrophobic solutes. From computations of site-site radial distribution function followed by analysis of coordination number, it is found that water molecules are tightly packed around the nonpolar particle at high pressure and the hydration number increases with increasing pressure. On the other hand, neopentane interacts preferentially with TMAO over water and although hydration of neopentane reduces in presence of this osmolyte, TMAO does not show any tendency to prevent the pressure-induced dispersion of neopentane moieties. It is also observed that TMAO molecules prefer a side-on orientation near the neopentane surface, allowing its oxygen atom to form favorable hydrogen bonds with water while maintaining some hydrophobic contacts with neopentane. Analysis of hydrogen-bond properties and solvation characteristics of TMAO reveals that TMAO can form hydrogen bonds with water and it reduces the identical nearest neighbor water molecules caused by high hydrostatic pressures. Moreover, TMAO enhances life-time of water-water hydrogen bonds and makes these hydrogen bonds more attractive. Implication of these results for counteracting effect of TMAO against protein denaturation at high pressures are discussed.     

Molecular dynamics simulation study on the inhibitory effects of choline-O-sulfate on hIAPP protofibrilation

Type 2 diabetes mellitus (T2Dm) is a neurodegenerative disease, which occurs due to the self-association of human islet amyloid polypeptide (hIAPP), also known as human amylin. It was reported experimentally that choline-O-sulfate (COS), a small organic molecule having a tertiary amino group and sulfate group, can prevent the aggregation of human amylin without providing the mechanism of the action of COS in the inhibition process. In this work, we investigate the influence of COS on the full-length hIAPP peptide by performing 500 ns classical molecular dynamics simulations. From pure water simulation (without COS), we have identified the residues 11–20 and 23–36 that mainly participate in the fibril formation, but in the presence of 1.07 M COS these residues become totally free of β-sheet conformation. Our results also show that the sulfate oxygen of COS directly interacts with the peptide backbone, which leads to the local disruption of peptide–peptide interaction. Moreover, the presence of favorable peptide-COS vdW interaction energy and high coordination number of COS molecules in the first solvation shell of the peptide indicates the hydrophobic solvation of the peptide residues by COS molecules, which also play a crucial role in the prevention of β-sheet formation. Finally, from the potential of mean force (PMFs) calculations, we observe that the free energy between two peptides is more negative in the absence of COS and with increasing concentration of COS, it becomes unfavorable significantly indicating that the peptide dimer formation is most stable in pure water, which becomes less favorable in the presence of COS. © 2019 Wiley Periodicals, Inc.     

Molecular dynamics simulations of the helical antimicrobial peptide ovispirin-1 in a zwitterionic dodecylphosphocholine micelle: insights into host-cell toxicity

We have carried out a 40-ns all-atom molecular dynamics simulation of the helical antimicrobial peptide ovispirin-1 (OVIS) in a zwitterionic diphosphocholine (DPC) micelle. The DPC micelle serves as an economical and effective model for a cellular membrane owing to the presence of a choline headgroup, which resembles those of membrane phospholipids. OVIS, which was initially placed along a micelle diameter, diffuses out to the water-DPC interface, and the simulation stabilizes to an interface-bound steady state in 40 ns. The helical content of the peptide marginally increases in the process. The final conformation, orientation, and the structure of OVIS are in excellent agreement with the experimentally observed properties of the peptide in the presence of lipid bilayers composed of 75% zwitterionic lipids. The amphipathic peptide binds to the micelle with its hydrophobic face buried in the micellar core and the polar side chains protruding into the aqueous phase. There is overwhelming evidence that points to the significant and indispensable participation of hydrophobic residues in binding to the zwitterionic interface. The simulation starts with a conformation that is unbiased toward the final experimentally known binding state of the peptide. The ability of the model to reproduce experimental binding states despite this starting conformation is encouraging.     

How does trimethylamine N-oxide counteract the denaturing activity of urea?

Trimethylamine N-oxide, TMAO, stabilizes globular proteins and is able to counteract the denaturing activity of urea. The mechanism of this counteraction has remained elusive up to now. A rationalization is proposed grounded on the same theoretical model used to clarify the origin of cold denaturation, and the denaturing activity of GdmCl versus the stabilizing one of Gdm(2)SO(4) [G. Graziano, Phys. Chem. Chem. Phys., 2010, 12, 14245-14252; G. Graziano, Phys. Chem. Chem. Phys., 2011, 13, 12008-12014]. The fundamental quantities are: (a) the difference in the solvent-excluded volume on passing from the N-state to the D-state, calculated in water and in aqueous osmolyte solution; (b) the difference in energetic attractions of the N-state and the D-state with the surrounding solvent molecules, calculated in water and in aqueous osmolyte solution. In aqueous 8 M urea + 4 M TMAO solution, the first quantity is so large and positive to counteract the second one that is large and negative due to preferential binding of urea molecules to the protein surface. This happens because aqueous 8 M urea + 4 M TMAO solution has a volume packing density markedly larger than that of water, rendering the cavity creation process much more costly. The volume packing density increase reflects the strength of the attractions of water molecules with both urea and TMAO molecules. This mechanism readily explains why TMAO counteraction is operative even though urea molecules are preferentially located on the protein surface.     

核糖核酸酶Sa表面水和尿素分子的分布和动力学行为的分子动力学模拟

利用分子动力学模拟研究了在不同尿素浓度下,核糖核酸酶Sa(RNase Sa)表面水和尿素分子的分布和动力学行为。 结果表明,尿素分子可与RNase Sa酶形成较强的相互作用,并取代其表面的水分子而富集在蛋白质表面。 尿素分子更倾向与RNase Sa酶的疏水残基作用,与RNase Sa酶主链形成氢键的能力更强。 尿素分子的平动和转动远远慢于水分子的平动和转动。 RNase Sa酶表面水分子的平动和转动随着尿素浓度增加而逐渐变慢,但RNase Sa酶表面尿素分子的动力学并不依赖于尿素浓度变化。 本研究中明晰的RNase Sa酶表面水和尿素分子分布和动力学有助于理解水和尿素分子对蛋白质稳定性的影响。