Thiazole‐Based γ‐Building Blocks as Reverse‐Turn Mimetic to Design a Gramicidin S Analogue: Conformational and Biological Evaluation

This paper describes the ability of a new class of heterocyclic γ‐amino acids named ATCs (4‐amino(methyl)‐1,3‐thiazole‐5‐carboxylic acids) to induce turns when included in a tetrapeptide template. Both hybrid Ac‐Val‐(R or S)‐ATC‐Ile‐Ala‐NH₂ sequences were synthesized and their conformations were studied by circular dichroism, NMR spectroscopy, MD simulations, and DFT calculations. It was demonstrated that the ATCs induced highly stable C₉ pseudocycles in both compounds promoting a twist turn and a reverse turn conformation depending on their absolute configurations. As a proof of concept, a bioactive analogue of gramicidin S was successfully designed using an ATC building block as a turn inducer. The NMR solution structure of the analogue adopted an antiparallel β‐pleated sheet conformation similar to that of the natural compound. The hybrid α,γ‐cyclopeptide exhibited significant reduced haemotoxicity compared to gramicidin S, while maintaining strong antibacterial activity.     

Do Valine Side Chains Have an Influence on the Folding Behavior of β‐Substituted β‐Peptides?

The influence of valine side chains on the folding/unfolding equilibrium and, in particular, on the 3₁₄‐helical propensity of β³‐peptides were investigated by means of molecular‐dynamics (MD) simulation. To that end, the valine side chains in two different β³‐peptides were substituted by leucine side chains. The resulting four peptides, of which three have never been synthesized, were simulated for 150 to 200 ns at 298 and 340 K, starting from a fully extended conformation. The simulation trajectories obtained were compared with respect to structural preferences and folding behavior. All four peptides showed a similar folding behavior and were found to predominantly adopt 3₁₄‐helical conformations, irrespective of the presence of valine side chains. No other well‐defined conformation was observed at significant population in any of the simulations. Our results imply that β³‐peptides show a structural preference for 3₁₄‐helices independent of the branching nature of the side chains, in contrast to what has been previously proposed on the basis of circular‐dichroism (CD) measurements.     

Enhanced sampling method in molecular simulations using genetic algorithm for biomolecular systems

We propose a molecular simulation method using genetic algorithm (GA) for biomolecular systems to obtain ensemble averages efficiently. In this method, we incorporate the genetic crossover, which is one of the operations of GA, to any simulation method such as conventional molecular dynamics (MD), Monte Carlo, and other simulation methods. The genetic crossover proposes candidate conformations by exchanging parts of conformations of a target molecule between a pair of conformations during the simulation. If the candidate conformations are accepted, the simulation resumes from the accepted ones. While conventional simulations are based on local update of conformations, the genetic crossover introduces global update of conformations. As an example of the present approach, we incorporated genetic crossover to MD simulations. We tested the validity of the method by calculating ensemble averages and the sampling efficiency by using two kinds of peptides, ALA3 and (AAQAA)₃. The results show that for ALA3 system, the distribution probabilities of backbone dihedral angles are in good agreement with those of the conventional MD and replica-exchange MD simulations. In the case of (AAQAA)₃ system, our method showed lower structural correlation of α-helix structures than the other two methods and more flexibility in the backbone ψ angles than the conventional MD simulation. These results suggest that our method gives more efficient conformational sampling than conventional simulation methods based on local update of conformations. © 2018 Wiley Periodicals, Inc.     

The Impact of Amino Acid Side Chain Mutations in Conformational Design of Peptides and Proteins

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N‐methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N‐methylation is often accompanied by considerable population of cis‐peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N‐methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.     

The Effect of Backbone Stereochemistry on the Folding of Acyclic β2, 3‐Aminoxy Peptides

As a new type of foldamer, β‐aminoxy peptides have the ability to adopt novel β N? O turns or β N? O helices in solution. Herein, we describe a new subclass of β‐aminoxy peptide, that is, peptides of acyclic β^{2, 3}‐aminoxy acids (NH₂OCHR₁CHR₂COOH), in which the presence of two chiral centers provides insight into the effect of backbone stereochemistry on the folding of β‐aminoxy peptides. Acyclic β^{2, 3}‐aminoxy peptides with syn and anti configurations have been synthesized and their conformations investigated by NMR, IR, and circular dichroism (CD) spectroscopic, and X‐ray crystallographic analysis. The β N? O turns or β N? O helices, which feature nine‐membered rings with intramolecular hydrogen bonds and have been identified previously in peptides of β³‐ and β^{2, 2}‐aminoxy acids, are also predominantly present in the acyclic β^{2, 3}‐aminoxy peptides with a syn configuration and N? O bonds gauche to the C_α? C_β bonds in both solution and the solid state. In the acyclic β^{2, 3}‐aminoxy peptides with an anti configuration, an extended strand (i.e., non‐hydrogen‐bonded state) is found in the solid state, and several conformations including non‐hydrogen‐bonded and intramolecular hydrogen‐bonded states are present simultaneously in nonpolar solvents. These results suggest that the backbone stereochemistry does affect the folding of the acyclic β^{2, 3}‐aminoxy peptides. Theoretical calculations on the conformations of model acyclic β^{2, 3}‐aminoxy peptides with different backbone stereochemistry were also conducted to elucidate structural characteristics. Our present work may provide useful guidelines for the design and construction of new foldamers with predicable structures.     

Computer Simulation of Long Side‐Chain Substituted Poly(phenylene vinylene) Polymers

A molecular dynamics (MD) simulation was employed to investigate structure features and segment orientation of four poly(phenylene vinylene) (PPV) derivatives with long, flexible side chains at room temperature. In the simulations, the main chains of the polymers were found to be semirigid and exhibit a tendency to coil into ellipsoidal helices or form zigzag conformations of limited regularity. The simulations show that continuous quasi‐coplanar segments along the backbone are in a range of ≈2–4 repeat units. The ordered orientation and coupling distance of interchain aromatic rings can be correlated with optical properties of materials. A simplified quantum‐mechanical method was developed to investigate optical properties based on MD trajectories. The method was tested to simulate the absorption spectra of four PPV derivatives. The absorption maxima of the calculated spectra are in reasonable agreement with experimental data. This work implies that long‐range electron transfer along the backbones of these polymers may not occur, but may be mediated by interchain interactions.     

Helical Folding of Conjugated Oligo(phenyleneethynylene): Chain‐Length Dependence,Solvent Effects,and Intermolecular Assembly

As a representative folding system that features a conjugated backbone, a series of monodispersed (o‐phenyleneethynylene)‐alt‐(p‐phenyleneethynylene) (PE) oligomers of varied chain length and different side chains were studied. Molecules with the same backbone but different side‐chain structures were shown to exhibit similar helical conformations in respectively suitable solvents. Specifically, oligomers with dodecyloxy side chains folded into the helical structure in apolar aliphatic solvents, whereas an analogous oligomer with tri(ethylene glycol) (Tg) side chains adopted the same conformation in polar solvents. The fact that the oligomers with the same backbone manifested a similar folded conformation independent of side chains and the nature of the solvent confirmed the concept that the driving force for folding was the intramolecular aromatic stacking and solvophobic interactions. Although all were capable of inducing folding, different solvents were shown to bestow slightly varied folding stability. The chain‐length dependence study revealed a nonlinear correlation between the folding stability with backbone chain length. A critical size of approximately 10 PE units was identified for the system, beyond which folding occurred. This observation corroborated the helical nature of the folded structure. Remarkably, based on the absorption and emission spectra, the effective conjugation length of the system extended more effectively under the folded state than under random conformations. Moreover, as evidenced by the optical spectra and dynamic light‐scattering studies, intermolecular association took place among the helical oligomers with Tg side chains in aqueous solution. The demonstrated ability of such a conjugated foldamer in self‐assembling into hierarchical supramolecular structures promises application potential for the system.     

Relating Trp‐Glu dipeptide fluorescence to molecular conformation: The role of the discrete chi 1 and chi 2 angles

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp‐Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time‐resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp‐Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles—60°, 180°, and 300°—play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH₃ cation‐π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp‐Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence. © 2013 Wiley Periodicals, Inc.     

Classical and quantum mechanical/molecular mechanical molecular dynamics simulations of alanine dipeptide in water: comparisons with IR and vibrational circular dichroism spectra

We have implemented the combined quantum mechanical (QM)/molecular mechanical (MM) molecular dynamics (MD) simulations of alanine dipeptide in water along with the polarizable and nonpolarizable classical MD simulations with different models of water. For the QM/MM MD simulation, the alanine dipeptide is treated with the AM1 or PM3 approximations and the fluctuating solute dipole moment is calculated by the Mulliken population analysis. For the classical MD simulations, the solute is treated with the polarizable or nonpolarizable AMBER and polarizable CHARMM force fields and water is treated with the TIP3P, TIP4P, or TIP5P model. It is found that the relative populations of right-handed alpha-helix and extended beta and P(II) conformations in the simulation trajectory strongly depend on the simulation method. For the QM/MM MD simulations, the PM3/MM shows that the P(II) conformation is dominant, whereas the AM1/MM predicts that the dominant conformation is alpha(R). Polarizable CHARMM force field gives almost exclusively P(II) conformation and other force fields predict that both alpha-helical and extended (beta and P(II)) conformations are populated with varying extents. Solvation environment around the dipeptide is investigated by examining the radial distribution functions and numbers and lifetimes of hydrogen bonds. Comparing the simulated IR and vibrational circular dichroism spectra with experimental results, we concluded that the dipeptide adopts the P(II) conformation and PM3/MM, AMBER03 with TIP4P water, and AMBER polarizable force fields are acceptable for structure determination of the dipeptide considered in this paper.     

The Coordination of NiII and CuII Ions to the Polyhistidyl Motif of Hpn Protein: Is It as Strong as We Think?

Hpn, one of Helicobacter pylori′s nickel‐accessory proteins, is an amazingly peculiar protein: Almost half of its sequence consists of polyhistidyl (poly‐His) residues. Herein, we try to understand the origin of this naturally occurring sequence, thereby shedding some light on the bioinorganic chemistry of Hpn′s numerous poly‐His repeats. By using potentiometric, mass spectrometric, and various spectroscopic techniques, we studied the Ni^II‐ and Cu^II complexes of the wild‐type Ac‐THHHHYHGG‐NH₂ fragment of Hpn and of its six analogues, in which consecutive residues (His or Tyr) were replaced by Ala (Ala‐substitution or Ala‐scan approaches), thereby resulting in Ac‐TAHHHYHGG‐NH₂, Ac‐THAHHYHGG‐NH₂, Ac‐THHAHYHGG‐NH₂, Ac‐THHHAYHGG‐NH₂, Ac‐THHHHAHGG‐NH₂, and Ac‐THHHHYAGG‐NH₂ peptides. We found that the His4 residue is critical for both Ni^II‐ and Cu^II‐ion binding and the effectiveness of binding varies even if the substituted amino acid does not take part in the direct binding interactions.     

Sialyldisaccharide conformations: a molecular dynamics perspective

Sialyldisaccharides are significant terminal components of glycoconjugates and their negative charge and conformation are extensively utilized in molecular recognition processes. The conformation and flexibility of four biologically important sialyldisaccharides [Neu5Acα(2-3)Gal, Neu5Acα(2-6)Gal, Neu5Acα(2-8)Neu5Ac and Neu5Acα(2-9)Neu5Ac] are studied using Molecular Dynamics simulations of 20 ns duration to deduce the conformational preferences of the sialyldisaccharides and the interactions which stabilize the conformations. This study clearly describes the possible conformational models of sialyldisaccharides deduced from 20 ns Molecular Dynamics simulations and our results confirm the role of water in the structural stabilization of sialyldisaccharides. An extensive analysis on the sialyldisaccharide structures available in PDB also confirms the conformational regions found by experiments are detected in MD simulations of 20 ns duration. The three dimensional structural coordinates for all the MD derived sialyldisaccharide conformations are deposited in the 3DSDSCAR database and these conformational models will be useful for glycobiologists and biotechnologists to understand the biological functions of sialic acid containing glycoconjugates.     

Efficient and clean charge derivatization of peptides for analysis by mass spectrometry

Charge derivatization with succinimidyloxycarbonylmethyl tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP‐Ac‐OSu) has great potential in several aspects of proteomics, such as peptide de novo sequencing, PTM analysis, etc. However, the excess reagent and its side products greatly limited its scope of use. Here, we report an improved method to perform charge derivatization of peptides by TMPP‐Ac‐OSu without interference from the excess reagent and corresponding side products. Briefly, the protein was first separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) or coagulated with the gel. The protein in‐gel was then incubated with a high concentration of reagent, followed by extensive washing. Afterwards, the protein was in‐gel digested with trypsin according to the routine protocol. The mainly resultant peptides were attached with one positive tag on the N‐termini or Lys‐ε‐NH₂. The process has been successfully applied to 2‐DE resolved protein spots. Compared to the native proteins, the derivatized counterparts have higher rates of PMF identification and more straightforward tandem mass spectra. This promising method should pave the way for the practical use of charge derivatization in proteomics. Copyright © 2010 John Wiley & Sons, Ltd.     

Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism

The four-stranded G-quadruplex motif is a conformation frequently adopted by guanine-rich nucleic acids that plays an important role in biology, medicine, and nanotechnology. Although vibrational spectroscopy has been widely used to investigate nucleic acid structure, association of particular spectral features with the quadruplex structure has to date been ambiguous. In this work, experimental IR absorption and vibrational circular dichroism (VCD) spectra of the model quadruplex systems d(G)(8) and deoxyguanosine-5'-monophosphate (5'-dGMP) were analyzed using molecular dynamics (MD) and quantum-chemical modeling. The experimental spectra were unambiguously assigned to the quadruplex DNA arrangement, and several IR and VCD bands related to this structural motif were determined. Involvement of MD in the modeling was essential for realistic simulation of the spectra. The VCD signal was found to be more sensitive to dynamical structural variations than the IR signal. The combination of the spectroscopic techniques with multiscale simulations provides extended information about nucleic acid conformations and their dynamics.     

2‐Methoxy‐4‐methylsulfinylbenzyl: A Backbone Amide Safety‐Catch Protecting Group for the Synthesis and Purification of Difficult Peptide Sequences

The use of 2‐methoxy‐4‐methylsulfinylbenzyl (Mmsb) as a new backbone amide‐protecting group that acts as a safety‐catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid‐mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi‐unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH₄I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the “difficult peptide” sequence H‐(Ala)₁₀‐NH₂ was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac‐(RADA)₄‐NH₂ sequence was chosen to validate this methodology. The improvements in solid‐phase peptide synthesis combined with the enhanced solubility of the quasi‐unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac‐(RADA)₄‐NH₂. To extend the scope of the approach, the challenging Aβ(1‐42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.     

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

Validation of the GROMOS 54A7 Force Field Regarding Mixed α/β‐Peptide Molecules

A molecular‐dynamics (MD) simulation study of two heptapeptides containing α‐ and β‐amino acid residues is presented. According to NMR experiments, the two peptides differ in dominant fold when solvated in MeOH: peptide 3 adopts predominantly β‐hairpin‐like conformations, while peptide 8 adopts a 14/15‐helical fold. The MD simulations largely reproduce the experimental data. Application of NOE atom? atom distance restraining improves the agreement with experimental data, but reduces the conformational sampling. Peptide 3 shows a variety of conformations, while still agreeing with the NOE and ³J‐coupling data, whereas the conformational ensemble of peptide 8 is dominated by one helical conformation. The results confirm the suitability of the GROMOS 54A7 force field for simulation or structure refinement of mixed α/β‐peptides in MeOH.     

Pi-helix preference in unsolvated peptides

Ion mobility measurements have been used to examine helix formations in the gas phase for a series of alanine/glycine-based peptides that incorporate a glutamic acid (E) and lysine (K) at various positions along the backbone. Incorporation of an EK pair lowers the percent helix for all positions (presumably because hydrogen bonding between the backbone and the E and K side chains stabilize the nonhelical globular conformations). The largest percent helix is found when the EK pair is in an i,i+5 arrangement, which suggests that the preferred helical conformation for these peptides is a pi-helix. This conclusion is supported by comparison of cross sections deduced from the ion-mobility measurements to average cross sections calculated for conformations obtained from molecular dynamics simulations. The glutamic acid and lysine may form an ion pair that is stabilized by interactions with the helix macro-dipole.     

Conformational preferences of an amyloidogenic peptide: IR spectroscopy of Ac-VQIVYK-NHMe

The (306)VQIVYK(311) sequence in the tau peptide is essential for the formation of intracellular amyloid fibrils related to Alzheimer's disease, where it forms interdigitating cross-beta-structures. The inherent conformational preferences of the capped hexapeptide Ac-VQIVYK-NHMe were characterized in the gas phase. IR/UV double-resonance spectroscopy of the peptide isolated in a cold molecular beam was used to probe the conformation of the neutral peptide. The influence of protonation at the lysine side chain was investigated at 298 K by characterizing the protonated peptide ion, Ac-VQIVYK(H(+))-NHMe, with IRMPD spectroscopy in the fingerprint and amide I/II band region in an FTICR mass spectrometer. The conformations for both neutral and protonated peptides were predicted by an extensive conformational search procedure followed by cluster analysis and then DFT calculations. Comparison of the experimental and computed IR spectra, with consideration of the relative energies, was used to assign the dominant conformations observed. The neutral peptide adopts a beta-hairpin-like conformation with two loosely extended peptide chains, demonstrating the preference of the sequence for extended beta-strand-like structures. In the protonated peptide, the lysine NH3(+) disrupts this extended conformation by binding to the backbone and induces a transition to a random-coil-like structure.