A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring hydrophobicity limits of polyproline helix with oligomeric octahydroindole‐2‐carboxylic acid

The polyproline‐II helix is the most extended naturally occurring helical structure and is widely present in polar, exposed stretches and “unstructured” denatured regions of polypeptides. Can it be hydrophobic? In this study, we address this question using oligomeric peptides formed by a hydrophobic proline analogue, (2S,3aS,7aS)‐octahydroindole‐2‐carboxylic acid (Oic). Previously, we found the molecular principles underlying the structural stability of the polyproline‐II conformation in these oligomers, whereas the hydrophobicity of the peptide constructs remains to be examined. Therefore, we investigated the octan‐1‐ol/water partitioning and inclusion in detergent micelles of the oligo‐Oic peptides. The results showed that the hydrophobicity is remarkably enhanced in longer oligomeric sequences, and the oligo‐Oic peptides with 3 to 4 residues and higher are specific towards hydrophobic environments. This contrasts significantly to the parent oligoproline peptides, which were moderately hydrophilic. With these findings, we have demonstrated that the polyproline‐II structure is compatible with nonpolar media, whereas additional manipulations of the terminal functionalities feature solubility in extremely nonpolar solvents such as hexane.     

Conformational preferences of a short Aib/Ala‐based water‐soluble peptide as a function of temperature

The amino acid Aib predisposes a peptide to be helical with context‐dependent preference for either 3₁₀‐ or α‐ or a mixed helical conformation. Short peptides also show an inherent tendency to be unfolded. To characterize helical and unfolded states adopted by water‐soluble Aib‐containing peptides, the conformational preference of Ac‐Ala‐Aib‐Ala‐Lys‐Ala‐Aib‐Lys‐Ala‐Lys‐Ala‐Aib‐Tyr‐NH₂ was determined by CD, NMR and MD simulations as a function of temperature. Temperature‐dependent CD data indicated the contribution of two major components, each an admixture of helical and extended/polyproline II structures. Both right‐ and left‐handed helical conformations were detected from deconvolution of CD data and ¹³C NMR experiments. The presence of a helical backbone, more pronounced at the N‐terminal, and a temperature‐induced shift in α‐helix/3₁₀‐helix equilibrium, more pronounced at the C‐terminal, emerged from NMR data. Starting from polyproline II, the N‐terminal of the peptide folded into a helical backbone in MD simulations within 5 ns at 60°C. Longer simulations showed a mixed‐helical backbone to be stable over the entire peptide at 5°C while at 60°C the mixed‐helix was either stable at the N‐terminus or occurred in short stretches through out the peptide, along with a significant population of polyproline II. Our results point towards conformational heterogeneity of water‐soluble Aib‐based peptide helices and the associated subtleties. The problem of analyzing CD and NMR data of both left‐ and right‐handed helices are discussed, especially the validity of the ellipticity ratio [θ]₂₂₂/[θ]₂₀₇, as a reporter of α‐/3₁₀‐ population ratio, in right‐ and left‐handed helical mixtures. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A comparison of the different helices adopted by ��- and ��-peptides suggests different reasons for their stability

The right‐handed α‐helix is the dominant helical fold of α‐peptides, whereas the left‐handed 3₁₄‐helix is the dominant helical fold of β‐peptides. Using molecular dynamics simulations, the properties of α‐helical α‐peptides and 3₁₄‐helical β‐peptides with different C‐terminal protonation states and in the solvents water and methanol are compared. The observed energetic and entropic differences can be traced to differences in the polarity of the solvent‐accessible surface area and, in particular, the solute dipole moments, suggesting different reasons for their stability.     

Length‐dependent stability of α‐helical peptide upon adsorption to single‐walled carbon nanotube

Earlier studies have shown that the helical content of α‐helical peptide decreases upon its interaction with carbon nanotube (CNT). Further, the length of the α‐helix varies from few residues in the small globular protein to several number of residues in structural and membrane proteins. In structural and membrane proteins, helices are widely present as the supercoil i.e., helical bundles. Thus, in this study, the length‐dependent interaction pattern of α‐helical peptides with CNT and the stability of isolated α‐helical fragment versus supercoiled helical bundle upon interaction with CNT have been investigated using classical molecular dynamics (MD) simulation. Results reveal that the disruption in the helical motif on interaction with CNT is directly proportional to the length of the helix. Also it is found that the shorter helix does not undergo noticeable changes in the helicity upon adsorption with CNT. On the other hand, helicity of longer peptides is considerably affected by its interaction with CNT. In contrast to the known fact that the stability of the helix increases with its length, the disruption in the helical peptide increases with its length upon its interaction with CNT. Comparison of results shows that structural changes in the isolated helical fragment are higher than that in supercoiled helix. In fact, helical chain in supercoiled bundle does not undergo significant changes in the helicity upon interaction with CNT. Both the length of the helical peptide and the inherent stability of the helical unit in the supercoiled helix influence the interaction pattern with the CNT. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 357–369, 2013.     

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2‐helix variants of PYY3‐36

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3‐36 is believed to perform its appetite‐suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3‐36 via its amphipathic α‐helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3‐36. We first studied membrane binding of PYY3‐36. Next, we designed a series of PYY3‐36 analogs to increase membrane‐binding affinity by substituting the N‐terminal segment with a de novo designed α‐helical, amphipathic sequence. These 2‐helix variants of PYY3‐36 were assembled by solid‐phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane‐bound state, which clearly showed that it formed the expected 2‐helix. The topology of the peptide–micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3‐36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2‐helix variant of PYY3‐36 will be useful as a tool compound for studying peptide–membrane interactions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

Structural characterization of a neurotoxic threonine‐rich peptide corresponding to the human prion protein α2‐helical 180–195 segment,and comparison with full‐length α2‐helix‐derived peptides

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrP^C full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrP^C makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of lipid‐binding properties of the N‐terminal helical segments in human apolipoprotein A‐I using fragment peptides

Although the N‐terminal region in human apolipoprotein (apo) A‐I is thought to stabilize the lipid‐free structure of the protein, its role in lipid binding is unknown. Using synthetic fragment peptides, we examined the lipid‐binding properties of the first 43 residues (1–43) of apoA‐I in comparison with residues 44–65 and 220–241, which have strong lipid affinity in the molecule. Circular dichroism measurements demonstrated that peptides corresponding to each segment have potential propensity to form α‐helical structure in trifluoroethanol. Spectroscopic and thermodynamic measurements revealed that apoA‐I (1–43) peptide has the strong ability to bind to lipid vesicles and to form α‐helical structure comparable to apoA‐I (220–241) peptide. Substitution of Tyr‐18 located at the center of the most hydrophobic region in residues 1–43 with a helix‐breaking proline resulted in the impaired lipid binding, indicating that the α‐helical structure in this region is required to trigger the lipid binding. In contrast, apoA‐I (44–65) peptide exhibited a lower propensity to form α‐helical structure upon binding to lipid, and apoA‐I (44–65/S55P) peptide exhibited diminished, but not completely impaired, lipid binding, suggesting that the central region of residues 44–65 is not pivotally involved in the formation of the α‐helical structure and lipid binding. These results indicate that the most N‐terminal region of apoA‐I molecule, residues 1–43, contributes to the lipid interaction of apoA‐I through the hydrophobic helical residues. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the free energy landscape of a model β‐hairpin peptide and its isoform

Secondary structural transitions from α‐helix to β‐sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. β‐hairpin peptides form basic components of anti‐parallel β‐sheets and are suitable model systems for characterizing the fundamental forces stabilizing β‐sheets in fibrillar structures. In this study, we explore the free energy landscape of the model β‐hairpin peptide GB1 and its E2 isoform that preferentially adopts α‐helical conformations at ambient conditions. Umbrella sampling simulations using all‐atom models and explicit solvent are performed over a large range of end‐to‐end distances. Our results show the strong preference of GB1 and the E2 isoform for β‐hairpin and α‐helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded β‐hairpin structures which differ from each other in the position of the β‐turn. We discuss the energetic factors contributing favorably to the formation of α‐helix and β‐hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non‐bonded interactions. Proteins 2014; 82:2394–2402. © 2014 Wiley Periodicals, Inc.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interruption of a 310‐helix by a single Gly residue in a poly‐Aib motif: A crystallographic study

The structural influence of a single Gly residue inserted into an Aib₁₆ homooligomer was studied in the solid state by X‐ray crystallography. The peptides N₃Aib₈GlyAib₈PheNH₂ ( 1 ) and CbzPheAib₈GlyAib₈ ( 2 ) were found to adopt well‐defined helical structures, which are broadly 3₁₀ helical. Indeed, 2 is the longest crystallographic 3₁₀ helix thus far reported. However, in the region of the central Gly residue, a loosening of the 3₁₀ structure is observed in both peptides, with 1 clearly showing local adoption of an α‐helical structure in the region of residues 7–9. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 62–69, 2011.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Structure of the hypothetical protein Ton 1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left‐handed helical turn in normal α‐solenoid protein

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α‐helices in a helix‐turn‐helix motif as a repeating unit, Ton1535 consists of right‐handed coiled N‐ and C‐terminal regions that are stacked together using helix bundles containing a left‐handed helical turn. One left‐handed helical turn in the right‐handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α‐helices on the convex surfaces of the N‐terminal region to the extended surface of the concave groove of the C‐terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.     

Phosphorylation stabilizes the N‐termini of α‐helices

The role of phosphorylation in stabilizing the N‐termini of α‐helices is examined using computer simulations of model peptides. The models comprise either a phosphorylated or unphosphorylated serine at the helix N‐terminus, followed by nine alanines. Monte Carlo/stochastic Dynamics simulations were performed on the model helices. The simulations revealed a distinct stabilization of the helical conformation at the N‐terminus after phosphorylation. The stabilization was attributable to favorable electrostatic interactions between the phosphate and the helix backbone. However, direct helix capping by the phosphorylated sidechain was not observed. The results of the calculations are consistent with experimental evidence on the stabilization of helices by phosphates and other anions. © 1999 John Wiley & Sons, Inc. Biopoly 49: 225–233, 1999     

Cosolvent,ions, and temperature effects on the structural properties of cecropin A‐Magainin 2 hybrid peptide in solutions

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A–Magainin 2. In TFE/water mixtures, the structure of the peptide retained α‐helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α‐helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N‐ter α‐helix segment during all the simulation. The α‐helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA‐MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 1–14, 2015.     

A strained DNA binding helix is conserved for site recognition,folding nucleation,and conformational modulation

Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 3₁₀ helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10⁴‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A dimer model of human calcitonin13‐32 forms an α‐helical structure and robustly aggregates in 50% aqueous 2,2,2‐trifluoroethanol solution

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13‐32 aggregated to a greater degree than native hCT under aqueous 2,2,2‐trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine‐T binding assays and atomic force microscopy suggest that the α‐helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β‐sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α‐helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.