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The bioavailability, release, and stability of pharmaceuticals under physicochemical conditions is the major cause of drug candidates failing during their clinical trials. Therefore, extensive efforts have been invested in the development of novel drug delivery systems that are able to transport drugs to a desired site and improve bioavailability. Hydrogels, and peptide hydrogels in particular, have been extensively investigated due to their excellent biocompatibility and biodegradability properties. However, peptide hydrogels often have weak mechanical strength, which limits their therapeutic efficacy. Therefore, a number of methods for improving their rheological properties have been established. This review will cover the broad area of drug delivery, focusing on the recent developments in this research field. We will discuss the variety of different types of nanocarrier drug delivery systems and then, more specifically, the significance and perspectives of peptide-based hydrogels. In particular, the interplay of intermolecular forces that govern the self-assembly of peptide hydrogels, progress made in understanding the distinct morphologies of hydrogels, and applications of non-canonical amino acids in hydrogel design will be discussed in more detail. [ABSTRACT FROM AUTHOR]

Methods for precisely constructing a β‐sheet assembly with number‐defined strands in solution remains quite limited due to its intense aggregation property. Here, we report the precise construction of a four‐stranded anti‐parallel β‐sheet by utilizing a non‐covalent approach. This was achieved by folding and assembly of Ag+ and a pentapeptide (1) with the Ala‐D3pa‐Gly‐3pa‐Val (3pa: β‐(3‐pyridyl)‐alanine) sequence, which was designed to form an interlocking Ag2(1)2 ring through metal cross‐linking of the side chains. NMR analyses and X‐ray crystallographic studies characterized the structure of the discrete β‐sheet assembly as well as the remarkable structural selectivity in terms of strands’ number, orientation and the sheet type. [ABSTRACT FROM AUTHOR]

The emergency of peptide-assembled nanomaterials motivates the efforts towards understanding the composition effects governing the assembly structure of peptides. Herein, we used time-lapse atomic force microscopy (AFM) to characterize the time-dependent structural transformation of phenylalanine (F)-rich self-assembled peptides and elucidated the impacts of composition heterogeneity on modulating peptide aggregation. Four binary peptides (F5Y5, F5A5, F5H5, and F5D5) were synthesized to arrange distinct types of amino acids, including aromatic tyrosine (Y), nonpolar alanine (A), cationic histidine (H), and anionic aspartic acid (D), in the proximity of an F-rich moiety. We compared the time-dependent structural transitions of these peptide assemblies using AFM. F5Y5 and F5A5 were observed to form fibril-like aggregates over time, whereas F5H5 and F5D5 assembled into globular particles during the time course examined. The impacts of neighboring amino acids on affecting F-rich peptide fibrillation are in line with the hydrophobicity scales of amino acid side chains. Specifically, Y and A facilitate the fibril aggregation, whereas H and D hinder the fibril formation of F-rich peptides. Our results manifest the hydrophobicity of amino acids proximal to the F residues is important for the fibril-like aggregation of peptides. [ABSTRACT FROM AUTHOR]

Integrated disease management and plant protection have been discussed with much fervor in the past decade due to the rising environmental concerns of using industrially produced pesticides. Members of the genus Trichoderma are a subject of considerable research today due to their several properties as biocontrol agents. In our study, the peptaibol production of Trichoderma longibrachiatum SZMC 1775, T. longibrachiatum f. bissettii SZMC 12546, T. reesei SZMC 22616, T. reesei SZMC 22614, T. saturnisporum SZMC 22606 and T. effusum SZMC 22611 were investigated to elucidate structure-activity relationships (SARs) between the properties of peptaibols and their 3D structures. The effects of peptaibol mixtures obtained from every Trichoderma strain were examined against nine commonly known bacteria. The lowest minimum inhibitory concentrations (MIC, mg ml−1) were exerted by T. longibrachiatum f. bissettii SZMC 12546 against Gram-positive bacteria, which was also able to inhibit the plant pathogenic Gram-negative Rhizobium radiobacter. Accelerated molecular dynamics (aMD) simulations were performed in aqueous solvent to explore the folding dynamics of 12 selected peptaibol sequences. The most characteristic difference between the peptaibols from group A and B relies in the ‘Gly-Leu-Aib-Pro’ and ‘Gly-Aib-Aib-Pro’ motifs (‘Aib’ stands for α-aminoisobutyric acid), which imparted a significant effect on the folding dynamics in water and might be correlated with their expressed bioactivity. In our aMD simulation experiments, Group A peptaibols showed more restricted folding dynamics with well-folded helical conformations as the most stable representative structures. This structural stability and dynamics may contribute to their bioactivity against the selected bacterial species.

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated. [ABSTRACT FROM AUTHOR]

Peptide T is a synthetic octapeptide fragment, which corresponds to the region 185–192 of the gp120 HIV coat protein and functions as a viral entry inhibitor. In this work, a folding molecular dynamics simulation of peptide T in a membrane‐mimicking (DMSO) solution was performed with the aim of characterizing the peptide's structural and dynamical properties. We show that peptide T is highly flexible and dynamic. The main structural characteristics observed were rapidly interconverting short helical stretches and turns, with a notable preference for the formation of β‐turns. The simulation also indicated that the C‐terminal part appears to be more stable than the rest of the peptide, with the most preferred conformation for residues 5–8 being a β‐turn. In order to validate the accuracy of the simulations, we compared our results with the experimental NMR data obtained for the T‐peptide in the same solvent. In agreement with the simulation, the NMR data indicated the presence of a preferred structure in solution that was consistent with a β‐turn comprising the four C‐terminal residues. An additional comparison between the experimental and simulation‐derived chemical shifts also showed a reasonable agreement between experiment and simulation, further validating the simulation‐derived structural characterization of the T‐peptide. We conclude that peptide folding simulations produce physically relevant results even when performed with organic solvents that were not part of the force field parameterization procedure. [ABSTRACT FROM AUTHOR]

pKa values of homorepeat hexapeptides with a 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) chromophore attached at the peptide C termini, through an asparagine derivative (Dbo), namely His6–Dbo (H6), Lys6–Dbo (K6), and Arg6–Dbo (R6), were determined by a novel fluorescence-based method. The fluorescence lifetime of Dbo in the peptides (τ) was measured as a function of pH. The side chains collide with Dbo intramolecularly and quench it efficiently only when they are deprotonated (i.e., pH ≥ side chain pKa). The pKa values of the H6, K6, and R6 peptides, attributable to side chain ionization, were found to be depressed compared to the pKa values of the His, Lys, and Arg residues in their free amino acid forms. We further looked into the structural changes of the peptides by molecular dynamics (MD) simulations; the peptides were structurally more expanded when their side chains are protonated. The structural expansion of the peptides reflects an electrostatic repulsion between the protonated side chain residues, which also accounts for the observed decrease in pKa values, which corresponds to a facilitated deprotonation, assisted by electrostatic repulsion. [ABSTRACT FROM AUTHOR]

Defensins are key components of both innate and adaptive immune responses to pathogens. Cryptdins are mouse alpha‐defensins that are secreted from Paneth cells in the small intestine and have disulfide‐stabilised structures and antibacterial activities against both Gram‐positive and Gram‐negative bacteria. The folding and three‐dimensional structures of alpha‐defensins are thought to depend on a conserved glycine residue that forms a β‐bulge. Here we investigated the role of this conserved glycine at position 19 of cryptdin‐4 (Crp4) in terms of the folding, structure and stability. A Crp4 variant with D‐Ala at position 19 folded efficiently, was stabilised by a large number of hydrogen bonds, and resisted proteolysis in simulated intestinal fluid. Although a variant with L‐Ala at position 19 was able to adopt the correct fold, it showed less efficient folding and was degraded more rapidly than the D‐Ala variant. These results demonstrate the key role that glycine residues can have in folding of bioactive peptides and can provide insights to guide design of stable antimicrobial peptides that fold efficiently. [ABSTRACT FROM AUTHOR]

The creation of artificial enzymes to mimic natural enzymes remains a great challenge owing to the complexity of the structural arrangement of the essential amino acids in catalytic centers. In this study, we used the phosphatase-based enzyme-instructed self-assembly (EISA) to supervise artificial esterases' final structures and catalytic activities. We reported that peptide precursors containing different phosphorylation sites could preorganize into alternated nanostructures and undergo dephosphorylation in the presence of alkaline phosphatase (ALP) with variation in kinetic and thermodynamic profiles. Although identical self-assembly compositions were formed after dephosphorylation, precursors with more enhanced preorganized states tended to better promote ALP dephosphorylation, facilitate further self-assembly, and strengthen the catalytic activities of the final assemblies. We envisioned that our strategy would be useful for further construction and manipulation of various artificial enzymes with superior catalytic activities. [ABSTRACT FROM AUTHOR]

Health care based on gene sequencing and genomics is increasingly becoming a reality: it is timely to review Crick’s sequence hypothesis for its fitness for this purpose. The sequence hypothesis is central to the prediction and correction of disease traits from gene sequence information. Considerable success in this respect has been achieved for rare diseases, but for the dominant part of the human disease burden, common diseases, little progress has been made since the completion of the sequencing of the human genome. It is argued here that the sequence hypothesis, namely the assumption that peptides will fold spontaneously to the native state protein, thus retaining the information coded in the originating genes, is not supported by a realistic physics-based assessment of the peptide to protein folding process.

The synthesis of hexa- and dodecapeptides functionalized with two Au(I)–phosphine complexes is reported. The high stability of the Au(I)–phosphine bond allowed orthogonal peptide-protecting-group chemistry, even when using hard Lewis acids like boron tribromide. This enabled the preparation of an Fmoc-protected lysine derivative carrying the Au(I) complex in a side chain, which was used in standard Fmoc-based solid-phase peptide synthesis protocols. Alanine and leucine repeats in the metallododecapeptide formed α-helical secondary structures in 2,2,2-trifluoroethanol–H2 O and 1,1,1,3,3,3-hexafluoroisopropanol–H2 O mixtures with high thermal stability, as shown by temperature-dependent CD spectroscopy studies. [ABSTRACT FROM AUTHOR]

Peptide flexibility is a determining factor in designing peptide-based drugs and in linker peptides. The flexibility is roughly inversely proportional to the size of the amino acid side chains in a peptide sequence. Glycine homo repeats are, therefore, the most flexible oligopeptides. We synthesized three oligopeptides: a relatively rigid peptide, His-(Arg)4-Trp (1), a flexible peptide, His-(Gly)4-Trp (2), and a "super-flexible" peptide; His-Gly-(GABA)-Gly-Trp (3) in which the central Gly-Gly unit in 2 was substituted by a γ-aminobutyric acid (GABA) linker. The only structural difference between 2 and 3 is that an amide bond in 2 is replaced by –CH2– units in 3. The frequency of end-to-end collisions, which serves as indicator of peptide flexibility, was measured fluorometrically. For comparing peptide flexibility, fluorescence emission spectra of their tryptophan residues were compared. Upon end-to-end collision, the N-terminal histidine residue efficiently quenches the fluorescence emission of the C-terminal tryptophan residue. The quenching rate is directly proportional to the peptide flexibility. The observed strongly increased flexibility in the γ-aminobutyric acid-containing peptide is due to the substitution of a single, rotationally restricted amide bond. Our result demonstrates the importance of amid bonds in limiting peptide dynamics. [ABSTRACT FROM AUTHOR]

Huntington disease is a neurodegenerative disease characterized by a polymorphic tract of polyglutamine repeats in exon 1 of the huntingtin protein, which is thought to be responsible for protein aggregation and neuronal death. The polyglutamine tract is preceded by a 17-residue sequence that is intrinsically disordered. This region is subject to phosphorylation, acetylation and other post-translational modifications in vivo, which modulate its secondary structure, aggregation and, subcellular localization. We used Molecular Dynamics simulations with a novel Hamiltonian-replica-exchange-based enhanced sampling method, SWISH, and an optimal combination of water and protein force fields to study the effects of phosphorylation and acetylation as well as cross-talk between these modifications on the huntingtin N-terminus. The simulations, validated by circular dichroism, were used to formulate a mechanism by which the modifications influence helical conformations. Our findings have implications for understanding the structural basis underlying the effect of PTMs in the aggregation and cellular properties of huntingtin.

Abstract The application of molecular dynamics simulations to study the folding and dynamics of peptides has attracted a lot of interest in the last couple of decades. Following the successful prediction of the folding of several proteins using molecular simulation, foldable peptides emerged as a favourable system mainly due to their application in improving protein structure prediction methods and in drug design studies. However, their performance is inherently linked to the accuracy of the empirical force fields used in the simulations, whose optimisation and validation is of paramount importance. Here we review the most important findings in the field of molecular peptide simulations and highlight the significant advancements made over the last twenty years. Special reference is made on the simulation of disordered peptides and the remaining challenge to find a force field able to describe accurately their conformational landscape. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Peptide epitopes mediate as many as 40% of protein-protein interactions and fulfill signaling, inhibition, and activation roles within the cell. Beyond protein recognition, some peptides can self- or coassemble into stable hydrogels, making them a readily available source of biomaterials. While these 3D assemblies are routinely characterized at the fiber level, there are missing atomistic details about the assembly scaffold. Such atomistic detail can be useful in the rational design of more stable scaffold structures and with improved accessibility to functional motifs. Computational approaches can in principle reduce the experimental cost of such an endeavor by predicting the assembly scaffold and identifying novel sequences that adopt said structure. Yet, inaccuracies in physical models and inefficient sampling have limited atomistic studies to short (two or three amino acid) peptides. Given recent developments in machine learning and advances in sampling strategies, we revisit the suitability of physical models for this task. We use the MELD (Modeling Employing Limited Data) approach to drive self-assembly in combination with generic data in cases where conventional MD is unsuccessful. Finally, despite recent developments in machine learning algorithms for protein structure and sequence predictions, we find the algorithms are not yet suited for studying the assembly of short peptides.

Health care based on gene sequencing and genomics is increasingly becoming a reality: it is timely to review Crick's sequence hypothesis for its fitness for this purpose. The sequence hypothesis is central to the prediction and correction of disease traits from gene sequence information. Considerable success in this respect has been achieved for rare diseases, but for the dominant part of the human disease burden, common diseases, little progress has been made since the completion of the sequencing of the human genome. It is argued here that the sequence hypothesis, namely the assumption that peptides will fold spontaneously to the native state protein, thus retaining the information coded in the originating genes, is not supported by a realistic physics-based assessment of the peptide to protein folding process. [ABSTRACT FROM AUTHOR]

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.

Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. [ABSTRACT FROM AUTHOR]

The villin headpiece helical subdomain (HP36) is one of the best known model systems for computational studies of fast-folding all-α miniproteins. HP21 is a peptide fragment-derived from HP36-comprising only the first and second helices of the full domain. Experimental studies showed that although HP21 is mostly unfolded in solution, it does maintain some persistent native-like structure as indicated by the analysis of NMR-derived chemical shifts. Here we compare the experimental data for HP21 with the results obtained from a 15-μs long folding molecular dynamics simulation performed in explicit water and with full electrostatics. We find that the simulation is in good agreement with the experiment and faithfully reproduces the major experimental findings, namely that (a) HP21 is disordered in solution with <10% of the trajectory corresponding to transiently stable structures, (b) the most highly populated conformer is a native-like structure with an RMSD from the corresponding portion of the HP36 crystal structure of <1 Å, (c) the simulation-derived chemical shifts-over the whole length of the trajectory-are in reasonable agreement with the experiment giving reduced χ2 values of 1.6, 1.4, and 0.8 for the Δδ13Cα, Δδ13CO, and Δδ13Cβ secondary shifts, respectively (becoming 0.8, 0.7, and 0.3 when only the major peptide conformer is considered), and finally, (d) the secondary structure propensity scores are in very good agreement with the experiment and clearly indicate the higher stability of the first helix. We conclude that folding molecular dynamics simulations can be a useful tool for the structural characterization of even marginally stable peptides. [ABSTRACT FROM AUTHOR]

Proteins are considered as the working force of cells. Their functionality is determined by their spatial form. In 1973 Anfinsen proposed that the spatial form is determined by the sequence of amino acids in the protein backbone. Yet, the number of possible sequences as well as the possible configurations is very large, making the task of predicting the protein's spatial form very difficult. Many approaches have been proposed, both classical and hybrid quantum – classical ones. We propose a novel hybrid algorithm. In our approach we utilized quantum walks, a proven model for universal quantum computation. We considered a simplified version of the protein backbone to be the evolution space of the quantum walk. The dihedral angles φ and ψ are introduced as phase factors to the quantum walk evolution. We also utilized a cost function to describe the system, where the R – chain, describing the specific amino acid, corresponds to a discrete value, affecting the cost functions value. Our aim is to minimize the cost function value, by updating the dihedral angles for specific regions of the Ramachandran plot, using a Metropolis algorithm. [Display omitted] [ABSTRACT FROM AUTHOR]

Protein–peptide interactions play essential functional roles in living organisms and their structural characterization is a hot subject of current experimental and theoretical research. Computational modeling of the structure of protein–peptide interactions is usually divided into two stages: prediction of the binding site at a protein receptor surface, and then docking (and modeling) the peptide structure into the known binding site. This paper presents a comprehensive CABS-dock method for the simultaneous search of binding sites and flexible protein–peptide docking, available as a user’s friendly web server. We present example CABS-dock results obtained in the default CABS-dock mode and using its advanced options that enable the user to increase the range of flexibility for chosen receptor fragments or to exclude user-selected binding modes from docking search. Furthermore, we demonstrate a strategy to improve CABS-dock performance by assessing the quality of models with classical molecular dynamics. Finally, we discuss the promising extensions and applications of the CABS-dock method and provide a tutorial appendix for the convenient analysis and visualization of CABS-dock results. The CABS-dock web server is freely available at http://biocomp.chem.uw.edu.pl/CABSdock/ . [ABSTRACT FROM AUTHOR]

Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges. [ABSTRACT FROM AUTHOR]

Short peptide helices have attracted attention as suitable building blocks for soft functional materials, but they are rarely seen in crystalline materials. A new artificial nanoassembly of short peptide helices in the crystalline state is presented in which peptide helices are arranged three-dimensionally by metal coordination. The folding and assembly processes of a short peptide ligand containing the Gly-Pro-Pro sequence were induced by silver(I) coordination in aqueous alcohol, and gave rise to a single crystal composed of polyproline II helices. Crystallographic studies revealed that this material possesses two types of unique helical nanochannel; the larger channel measures more than 2 nm in diameter. Guest uptake properties were investigated by soaking the crystals in polar solutions of guest molecules; anions, organic chiral molecules, and bio-oligomers are effectively encapsulated by this peptide-folded porous crystal, with moderate to high chiral recognition for chiral molecules. [ABSTRACT FROM AUTHOR]

Abstract: Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated. [Copyright &y& Elsevier]

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains. [ABSTRACT FROM AUTHOR]

The detailed behavior of many molecular processes in the cell, such as protein folding, protein complex assembly, and gene regulation, transcription and translation, can often be accurately captured by stochastic chemical kinetic models. We investigate a novel computational problem involving these models---that of finding the most probable sequence of reactions that connects two or more states of the system observed at different times. We describe an efficient method for computing the probability of a given reaction sequence, but argue that computing most probable reaction sequences is EXPSPACE-hard. We develop exact (exhaustive) and approximate algorithms for finding most probable reaction sequences. We evaluate these methods on test problems relating to a recently-proposed stochastic model of folding of the Trp-cage peptide. Our results provide new computational tools for analyzing stochastic chemical models, and demonstrate their utility in illuminating the behavior of real-world systems.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/G i pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases • We still lack selective pharmacological tools for the muscarinic receptor subtypes. • MT9 was identified from the black mamba venom and synthesized. • MT9 was synthesized and 3D structure determined, showing it is a member of the three-finger toxins. • It acts at low nM potency as a selective non-competitive antagonist of M2 receptor. • MT9 holds promises for the treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Fold me to hold me: A specific binding agent against the delta‐like ligand 4 protein, which is expressed in angiogenesis, was identified by ribosome display. Specific tumor binding of this molecular imaging agent depends on the appropriate disulfide connectivity of its scaffold. [ABSTRACT FROM AUTHOR]

To determine local chirality driven conformational preferences of small aminocyclobutane-1-carboxylic acid derivatives, X-(ACBA)-Y, their matrix-isolation IR spectra were recorded and analyzed. For the very first time model systems of this kind were deposited in a frozen (~10 K) noble gas matrix to reduce line width and thus, the recorded sharp vibrational lines were analyzed in details. For cis- (S,R)- 1 monomer two ' zigzag' conformers composed of either a six or an eight-membered H-bonded pseudo ring was identified. For trans- (S,S)- 2 stereoisomer a zigzag of an eight-membered pseudo ring and a helical building unit were determined. Both findings are fully consistent with our computational results, even though the relative conformational ratios were found to vary with respect to measurements. For the dimers ( S,R,S,S)- 3 and ( S,S,S,R)- 4 as many as four different cis,trans and three different trans,cis conformers were localized in their matrix-isolation IR (MI-IR) spectra. These foldamers not only agree with the previous computational and NMR results, but also unambiguously show for the first time the presence of a structure made of a cis,trans conformer which links a ' zigzag' and a helical foldamer via a bifurcated H-bond. The present work underlines the importance of MI-IR spectroscopy, applied for the first time for triamides to analyze the conformational pool of small biomolecules. We have shown that the local chirality of a β-amino acid can fully control its backbone folding preferences. Unlike proteogenic α-peptides, β- and especially (ACBA) type oligopeptides could thus be used to rationally design and influence foldamer's structural preferences. [ABSTRACT FROM AUTHOR]

In the last decade many advances have been made on molecular dynamics simulations and different force fields were developed from the combination of differentiable functions of the atomic coordinates to represent the system energy and of parameters that describe the geometric and energetic properties of inter-particle interactions. However, it has been shown that very subtle modifications to commonly used molecular mechanical potentials can significantly alter the behavior of those potentials inducing stabilizing or destabilizing effects in the patterns of peptides or proteins. In this article we describe the behavior of polyalanine peptides under the influence of various 'force fields'. The polyalanines were chosen as study model since their structural features were already studied experimentally and thus our computational results were easily comparable with the experimental ones. In particular, three peptides composed of 8, 10 and 12 alanine residues were subjected to molecular dynamics simulations using 12 different force fields to understand what is the most appropriate force field to properly simulate their folding. Our results showed that Amber99ϕ is the best force field able to generate helical conformations in agreement with experimental data. [ABSTRACT FROM AUTHOR]

Abstract: Folding simulations of a choline-binding peptide derived from the Streptococcus pneumoniae LytA protein converged to a model of the peptide''s folded state structure which is in outstanding agreement with the experimentally-determined structures, reaching values for the root mean squared deviation as low as 0.24Å for the peptide''s backbone atoms and 0.65Å for all non-hydrogen atoms. [Copyright &y& Elsevier]

Considering N-methylacetamide (NMA) as a model compound, new interaction parameters are developed for the amide function in the GROMOS force field that are compatible with the recently derived 53A6OXY parameter set for oxygen-containing chemical functions. The resulting set, referred to as 53A6OXY+A, represents an improvement over earlier GROMOS force-field versions in the context of the pure-liquid properties of NMA, including the density, heat of vaporization, dielectric permittivity, self-diffusion constant and viscosity, as well as in terms of the Gibbs hydration free energy of this molecule. Assuming that NMA represents an adequate model compound for the backbone of peptides, 53A6OXY+A may be expected to also provide an improved description of polypeptide chains. As an initial test, simulations are reported for two β-peptides characterized by very different folding properties in methanol. For these systems, earlier force-field versions provided good agreement with experimental NMR data, and the test shows that the improved description achieved in the context of NMA is not accompanied by any deterioration in the representation of the conformational properties of these peptides. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The pHLIP peptide has three states: (I) soluble in aqueous buffer, (II) bound to the bilayer surface at neutral pH, and (III) inserted as a transmembrane (TM) helix at acidic pH. The membrane insertion of pHLIP at low pH can be used to target the acidic tissues characteristic of different diseases, such as cancer. We find that the a-helix content of state II depends on lipid acyl chain length but not cholesterol, suggesting the helicity of the bound state may be controlled by the bilayer elastic bending modulus. Experiments with the P20G variant show the proline residue in pHLIP reduces the a-helix content of both states II and III. We also observe that the membrane insertion pKa is influenced by membrane physical properties, following a biphasic pattern similar to the membrane thickness optima observed for the function of eukaryotic membrane proteins. Because tumor cells exhibit altered membrane fluidity, we suggest this might influence pHLIP tumor targeting. We used a cell insertion assay to determine the pKa in live cells, observing that the properties in liposomes held in the more complex plasma membrane. Our results show that the formation of a TM helix is modulated by both the conformational propensities of the peptide and the physical properties of the bilayer. These results suggest a physical role for helix-membrane interactions in optimizing the function of more complex TM proteins. [ABSTRACT FROM AUTHOR]

Abstract: We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides. [Copyright &y& Elsevier]

Folding and unfolding of β-peptides has been studied extensively by molecular dynamics (MD) simulation in the past decade. In these simulations, a non-polarizable model for the solvent (mostly methanol) was used. This work has investigated the effect of using a polarizable methanol solvent model upon the folding equilibrium of β-peptides. Thirteen MD simulations covering a total simulation length of 1.25 µs for three differently folding β-peptides were analyzed. The agreement with experimental data was slightly improved by applying the polarizable solvent. In the polarizable solvent, helical structures, which have a large dipole moment, are stabilized, while no obvious effect was detected in the simulations of peptides that have a hairpin structure as the dominant fold. The introduction of electronic polarizability into the solvent model appears of importance to a proper description of folding equilibria if these are determined by competing solute conformations that have different dipole moments. [ABSTRACT FROM AUTHOR]

Computer simulation using molecular dynamics is increasingly used to simulate the folding equilibria of peptides and small proteins. Yet, the quality of the obtained results depends largely on the quality of the force field used. This comprises the solute as well as the solvent model and their energetic and entropic compatibility. It is, however, computational very expensive to perform test simulations for each combination of force-field parameters. Here, we use the one-step perturbation technique to predict the change of the free enthalpy of folding of a β-peptide in methanol solution due to changing a variety of force-field parameters. The results show that changing the solute backbone partial charges affects the folding equilibrium, whereas this is relatively insensitive to changes in the force constants of the torsional energy terms of the force field. Extending the cut-off distance for nonbonded interactions beyond 1.4 nm does not affect the folding equilibrium. The same result is found for a change of the reaction-field permittivity for methanol from 17.7 to 30. The results are not sensitive to the criterion, e.g., atom-positional RMSD or number of hydrogen bonds, that is used to distinguish folded and unfolded conformations. Control simulations with perturbed Hamiltonians followed by backward one-step perturbation indicated that quite large perturbations still yield reliable results. Yet, perturbing all solvent molecules showed where the limitations of the one-step perturbation technique are met. The evaluated methodology constitutes an efficient tool in force-field development for molecular simulation by reducing the number of required separate simulations by orders of magnitude. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010 [ABSTRACT FROM AUTHOR]

Abstract: Cryptochromes (CRY) and photolyases (PL) use a common flavin adenine dinucleotide cofactor and homologous protein scaffold to accomplish numerous, seemingly dissimilar functions. PL repairs UV-damaged DNA in a mechanism requiring light and DNA base flipping. CRY cannot repair DNA, and instead function in core biological processes including plant photomorphogenesis, circadian rhythm, and magnetoreception. One subclass, CRY-DASH, does catalyze repair of single-stranded DNA; compromised base flipping may deactivate its tight binding to duplex DNA substrates. We recently demonstrated that the a “recognition loop” involved in DNA binding by both PL and CRY-DASH is among the most flexible regions in the two proteins, and exhibits especially heightened dynamics in CRY-DASH. Here, we establish that these distinct dynamics are encoded by the loop sequences: we quantify the flexibility of the isolated loop peptides through the kinetics and activation parameters for their folding. Mirroring the dynamics within the proteins, the CRY-DASH recognition loop peptide folds 2.5-fold faster than its counterpart in PL, predominantly due to a lower enthalpy of activation. We propose that these distinct dynamics are functionally significant in DNA recognition. Binding duplex DNA in the catalytically-active base-flipped conformation imposes significant order on the recognition loop, and a corresponding entropic penalty. This may be surmounted by the more preorganized PL recognition loop, but may impose too large a barrier for the more dynamic loop in CRY-DASH. These results suggest that evolution of protein dynamics, through local sequence tuning in the recognition loop, may be an important mechanism for functional diversification in PL and CRY. [Copyright &y& Elsevier]

Abstract: We demonstrate in this work that scanning tunneling microscopy (STM) provides a useful approach to obtaining structural information about human islet amyloid polypeptide (hIAPP) and rat islet amyloid polypeptide (rIAPP) assembly on highly oriented pyrolytic graphite (HOPG) with sub-molecular resolution. The observed hIAPP and rIAPP lamellae consisted of parallel stripes. The STM images of hIAPPs show multiple molecular folding structures, with an average of 11 amino acid residues for the core regions. In addition, the STM images also reveal the assembly characteristics of rIAPP lamellae and may indicate a secondary structural conformation from random coil to beta-sheet-like on the graphite surface. [Copyright &y& Elsevier]

Abstract: Molecular dynamics simulated annealing (SA-MD) simulations are frequently used for refinement and optimization of peptide and protein structures. Depending on the simulation conditions and simulation length SA-MD simulations can be trapped in locally stable conformations far from the global optimum. As an alternative replica exchange molecular dynamics (RexMD) simulations can be used which allow exchanges between high and low simulation temperatures at all stages of the simulation. A significant drawback of RexMD simulations is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A combined SA-MD and RexMD approach termed SA-RexMD is suggested that employs a small number of replicas (4) and starts initially with a set of high simulation temperatures followed by gradual cooling of the set of temperatures until a target temperature has been reached. The protocol has been applied for the folding of several peptide systems and for the refinement of protein model structures. In all the cases, the SA-RexMD method turned out to be significantly more efficient in reaching low energy structures and also structures close to experiment compared to continuous MD simulations at the target temperature and to SA-MD simulations at the same computational demand. The approach is well suited for applications in structure refinement and for systematic force field improvement. [Copyright &y& Elsevier]

Abstract: Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a β-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0–0.26mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065mol/L), the peptide folds faster than that in water, but it cannot fold to the β-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose. [Copyright &y& Elsevier]

We have investigated the site-specific folding kinetics of a photo-switchable cross-linked α-helical peptide by using single 13C = 18O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6°C), whereas at high temperatures (45°C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide. and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a, stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained α-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an α-helical segment within a protein, rather than an isolated α-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein. [ABSTRACT FROM AUTHOR]

Calculations of the electronic structure of solids began decades ago, but only recently have solid-state quantum techniques become sufficiently reliable that their application is nearly as routine as quantum chemistry is for molecules. We aim to introduce chemists to the pros and cons of first-principles methods that can provide atomic-scale insight into the properties and chemistry of bulk materials, interfaces, and nanostructures. The techniques we review include the ubiquitous density functional theory (DFT'), which is often suffi- cient, especially for metals; extensions such as DFT + Uand hybrid DFT, which incorporate exact exchange to rid DFT' of its spuri- ous self-interactions (critical for some semiconductors and strongly correlated materials); many-body Green's function (GWand Bethe- Salpeter) methods for excited states; quantum Monte Carlo, in prin- ciple an exact theory but for which forces (hence structure optimization and dynamics) are problematic; and embedding theories that locally refine the quantum treatment to improve accuracy. [ABSTRACT FROM AUTHOR]

A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the β-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 üs. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the β-strand. [ABSTRACT FROM AUTHOR]

Abstract: The secondary structure of two synthetic peptides from heptad-repeat domains of herpes simplex virus 1 glycoprotein H was determined by circular dichroism. In particular, the propensity of these peptides to assume an ordered structure was investigated upon by changing the solvent''s polarity and the temperature. A reduction of solvent polarity led to a significant increase in the α-helix content in the case of HR1, whereas only a slight change in the secondary structure was observed in the case of HR2. In both cases the conformational change followed a two-state transition model. The interaction of the peptides was monitored by the conformational change in the mixture with respect to the single peptides. However, formation of the complex did not significantly enhance thermal stability. A reliable estimation of the secondary structure was obtained by optimising the experimental conditions to collect CD data down to 180 nm, and by comparing the structure data yielded by different software packages. [Copyright &y& Elsevier]