Chiral, fully extended helical peptides

The synthesis of the N-protected (blocked) homo-peptide esters from the chiral C(α)-ethyl, C(α)-n-pentylglycine was performed in solution to the hexapeptide level. The conformational propensity exhibited by these oligomers in chloroform solution and in the crystal state was assessed by use of FTIR absorption, NMR, and X-ray diffraction. The results indicated that fully extended helical structures (2.0(5)-helices) are overwhelmingly adopted irrespective of the peptide main-chain length. This oligomeric series is of great interest as it is characterized by the longest C ( i )(α) ,…, C ( i+1 )(α) (per residue) separation achievable in the class of chiral, rigid, helical peptide spacers based on α-amino acids.     

Circular dichroism studies on helical structure preferences of amino acid residues of proteins caused by sodium dodecyl sulfate

The extent of helical structure of 19 intact proteins and of 15 proteins with no disulfide bridges in the absence and presence of 10 mM sodium dodecyl sulfate (SDS) was determined using the curve-fitting method of circular dichroic spectra. The change in helicity caused by the addition of SDS was examined as a function of each amino acid fraction. An increase in the helicity upon the addition of SDS occurred in most of the proteins with no disulfide bridges (C proteins) and containing more than 0.06 Lys fraction. In most of the intact proteins (B proteins), most of which contained disulfide bridges, helicity in SDS decreased with an increase in Lys fraction. The helicity of the C proteins in SDS also tended to increase with an increase in the Leu and Phe fractions, while it decreased with an increase in the Gly fraction. For the helicity of the B proteins in SDS, there was a tendency to increase with increased Asn fraction and decrease with increased His fraction. On the other hand, amino acids were divided into eight groups according to their side-chain properties and the conformational preference for each of the amino acid groups of C proteins was calculated using a simple assumption.     

Chiral recognition in the binding of helicenediamine to double strand DNA: interactions between low molecular weight helical compounds and a helical polymer

Binding of a helicene, 5,8-bis(aminomethyl)-1,12-dimethylbenzo[c]phenanthrene, to calf thymus DNA was studied using UV, CD, and fluorescence spectroscopy as well as calorimetry. The enantiomeric helicenes strongly bound to the double strand DNA possessing the right-handed helical structure. In addition, chiral recognition was observed in the binding, where the (P)-helicene with the right-handed helicity formed more stable complex than the (M)-helicene with the left-handed helicity. The binding studies of the helicenes and natural nucleosides by 1H NMR spectroscopy also revealed the higher affinity to the (P)-helicene. Both monomeric and polymeric nucleic acids thus turned out to favor the (P)-helicity.     

Triple helical stabilities of guest-host collagen mimetic structures

The peptoid Nleu (N-isobutylglycine) has been successfully incorporated into a series of collagen mimetics composed of Gly-Pro-Nleu and Gly-Nleu-Pro sequences and has been able to maintain triple helices in appropriate structures. The achiral trimeric sequence Gly-Nleu-Nleu as a guest sequence in structures such as Ac-(Gly-Pro-Hyp)3-(Gly-Nleu-Nleu)3-(Gly-Pro-Hyp)3-NH2 retains triple helicity. As an extension of this study, we report, in this paper, on a series of guest-host collagen mimetic structures in which Gly-Nleu-Pro sequences are employed as the host. The guest sequences for these guest-host structures include Gly-Nleu-Nleu and Gly-Nx-Pro sequences where Nx is composed of a variety of alkyl and aralkyl peptoid residues. From these guest-host collagen mimetic structures, we are able to elucidate the contributions of hydrophobic and steric effects on triple helix formation. The Gly-Nleu-Pro sequences have been shown to be effective in inducing triple helicity. Conformational characterization of the guest-host collagen mimetic structures was established by techniques such as temperature-dependent optical rotation measurements and circular dichroism (CD) spectroscopy.     

Monte Carlo folding of trans-membrane helical peptides in an implicit generalized Born membrane

An efficient Monte Carlo (MC) algorithm using concerted backbone rotations is combined with a recently developed implicit membrane model to simulate the folding of the hydrophobic transmembrane domain M2TM of the M2 protein from influenza A virus and Sarcolipin at atomic resolution. The implicit membrane environment is based on generalized Born theory and has been calibrated against experimental data. The MC sampling has previously been used to fold several small polypeptides and been shown to be equivalent to molecular dynamics (MD). In combination with a replica exchange algorithm, M2TM is found to form continuous membrane spanning helical conformations for low temperature replicas. Sarcolipin is only partially helical, in agreement with the experimental NMR structures in lipid bilayers and detergent micelles. Higher temperature replicas exhibit a rapidly decreasing helicity, in agreement with expected thermodynamic behavior. To exclude the possibility of an erroneous helical bias in the simulations, the model is tested by sampling a synthetic Alanine-rich polypeptide of known helicity. The results demonstrate there is no overstabilization of helical conformations, indicating that the implicit model captures the essential components of the native membrane environment for M2TM and Sarcolipin.     

Structural effects of extracellular loop mutations in CFTR helical hairpins

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations – glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.     

Turn and helical peptide spacers: combined distance and angular dependencies in the exciton-coupled circular dichroism of intramolecularly interacting bis-porphyrins

In a previous study we examined by the exciton-coupled circular dichroic method the distance effect generated by three-rigid-turn and helical-peptide spacers. In this connection porphyrins were confirmed to be excellent reporter chromophores. In the present investigation we have completed this research by expanding the original analysis to the assessment of the combined role of the chromophore distance and orientation with use of the same porphyrin derivatives and additional four analogous spacers of different main-chain lengths. We find that not only the intramolecular separation of the chromophores, but the angular dependence between the directions of their effective transition moments as well, are responsible for the onset and modulation of the intensity of the exciton-coupling phenomenon of the porphyrin Soret band.     

Temperature-dependent optical rotatory dispersion properties of helical muscle proteins and homopolymers

Thermally induced helix–coil transitions of myosin rod, light meromyosin, and tropomyosin were studied by optical rotatory dispersion (ORD). Fractional helicity was calculated from both the Moffitt-Yang parameter, b₀, and the corrected mean residue rotation [m′] at 231.4 nm. Between 3 and 30°C, [m′] increases linearly with a slope of 59/°C, whereas b₀ is virtually constant, indicating apparently different thermal melting behavior. Poly(L -lysine) and poly(L -glutamic acid) in their helical forms and myoglobin also show a nearly linear temperature dependence of [m′]_231.4. Muscle proteins in 6M guanidine hydrochloride and the random-coil forms of the homopolymers exhibit temperature-dependent values of [m′]_231.4 and b₀. We conclude from these observations that ORD properties of both α-helices and random-coil polypeptides have significant intrinsic temperature dependencies. A new method of estimating fractional helicity as a function of temperature is proposed.     

Molecular spacers for physicochemical investigations based on novel helical and extended peptide structures

A proper understanding of the detailed nature and mechanism of physicochemical interactions depends heavily upon our ability to design and synthesize conformationally constrained 3D structures whose intercomponent geometry (either rigorously rigid or able to undergo destructuration, if required, but in all cases precisely tunable) would be well defined. To this end we have recently reported a few initial studies and we are currently working on the exploitation of stable, short, helical peptide spacers based on achiral and/or chiral Calpha-tetrasubstituted alpha-amino acids. These building blocks are known to force the peptides either to predominantly fold into a 3(10)-helical structure or to adopt a fully extended, planar 2.0(5)-helix. The systems under investigation involve a donor and an acceptor moiety linked to the N- and C-termini of the oligopeptide spacer main chain. By increasing the number of intervening residues the donor.acceptor separation can be easily modulated. This review highlights details of these two novel peptide secondary structures and their use as molecular spacers in physicochemical investigations.     

Effect of osmotic potential, pH, and temperature on the growth of a helical, motile mycoplasma causing corn stunt disease.

Growth characteristics of corn stunt spiroplasma, a helical, motile mycoplasma, were studied over a range of osmolality, pH, and temperature in a simple medium containing 20% (v/v) agamma horse serum, 1.5% (w/v) PPLO broth, and various concentrations of sucrose. The spiroplasma was able to grow in a wide spectrum of osmolalities from 360 to 1120 mosm. Optimal growth was observed in media that contained 0.25-0.35 M sucrose. The organism became longer and thinner in media adjusted to 0.65 M sucrose or more. The spiroplasma lost helicity and motility immediately after transfer to media at pH 5.4 or lower. Optimal pH for growth was 7.2. No growth was observed at pH lower than 5.4 or higher than 8.0. Optimal temperature for growth was 32 degrees C. Very little or no growth was observed at temperatures lower than 15 degrees C or higher than 35 degrees C.     

Analysis of the segmental stability of helical peptides by isotope-edited infrared spectroscopy

Isotope-edited infrared spectroscopy has the ability to probe the segmental properties of long biopolymers. In this work, we have compared the infrared spectra of a model helical peptide ((12)C) Ac-W-(E-A-A-A-R)(6)-A-NH(2), described originally by Merutka et al. (Biochemistry 1991;30:4245-4248) and three derivatives that are (13)C labeled at the backbone carbonyl of alanines. The locations of six isotopically labeled alanines are at the N-terminal, C-terminal, and the middle two repeating units of the peptide. Variation in temperature from 1 degrees to 91 degrees C transformed the peptides from predominantly helical to predominantly disordered state. Amplitude and position of the infrared amide I' absorption bands from (12)C- and (13)C-labeled segments provided information about the helical content. Temperature dependence of infrared spectra was used to estimate segmental stability. As a control measure of overall peptide stability and helicity (independent of labeling), the temperature dependence of circular dichroism spectra in the far-UV range at identical conditions (temperature and solvent) as infrared spectra was measured. The results indicate that the central quarter of the 32 amino acids helix has the maximal helicity and stability. The midpoint of the melting curve of the central quarter of the helix is 5.4 +/- 0.8 degrees C higher than that of the termini. The N-terminal third of the helix is more helical and is 2.0 +/- 1.4 degrees C more stable than the C-terminus.     

Thioether side chain cyclization for helical peptide formation: inhibitors of estrogen receptor-coactivator interactions.

Cystine, lanthionine, and cystathionine containing cyclic peptides incorporating the signature nuclear receptor (NR) box (LXXLL) motif have been synthesized and the abilities of these peptides to inhibit estrogen receptor (ER)-coactivator interactions have been determined. We found that helicity of these peptides directly correlated with their bioactivity. Cystathionine proved to be a redox-stable, isosteric replacement for the cystine disulfide. Cystathionine containing peptide 3 showed higher helical character and a lower inhibition constant (Ki, 7 nm) when compared with its cystine counterpart.     

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such ‘normalization’ to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.     

Amphipathic alpha helical antimicrobial peptides.

Antimicrobial peptides (AMPs) that assume an amphipathic alpha helical structure are widespread in nature. Their activity depends on several parameters including the sequence, size, degree of structure formation, cationicity, hydrophobicity and amphipathicity. The analysis of numerous natural AMPs provided representative values for these parameters and led to a sequence template with which to generate potent artificial lead AMPs. Sequences were then varied in a rational manner, using both natural and nonproteinogenic amino acids, to probe the individual roles of each parameter in modulating biological activity. A high cationicity combined with a stabilized amphipathic alpha helical structure conferred enhanced cidal activity towards all the cell types considered, and was a requirement for Gram-positive bacteria and fungi. An elevated helicity also correlated with increased hemolytic activity. The structural requirements for activity against several Gram-negative bacteria were instead considerably less stringent, so that it persisted in peptides in which formation of a helical structure and/or amphipathicity were impeded. Either a reduced charge or a reduced hydrophobicity resulted in generally inactive peptides. These observations, combined with the kinetics of bacterial membrane permeabilization and time-killing are discussed in terms of currently accepted models of action for this type of peptide. The simple guidelines obtained in this study allowed the design of highly active shortened AMPs and may be generally useful in the development of this type of peptides as anti-infective agents.     

Distance dependency of exciton coupled circular dichroism using turn and helical peptide spacers

Porphyrins are promising chromophores for the investigation of the still unexplored area of 3-dimensional structural studies of proteins by using the exciton coupled circular dichroism (CD) method. The synthesis, conformational characterization by FTIR absorption and (1)H-NMR, and CD properties are described for a model bis-porphyrin system based on homooligo-[L-(alphaMe)Val](n) peptides as rigid spacers. In particular, the coupled CD phenomenon is experimentally detected, the intensity of which is modulated by the interchromophoric distance. These results extend and integrate those already reported with steroid, dimeric steroid, and brevetoxin bridges.     

Nonnative helical motif in a chaperone-bound protein fragment

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb_1-77) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-β, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-β-bound apoMb_1-77 displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.     

Mechanistic insight into the physiological relevance of helical blood flow in the human aorta: an in vivo study

The hemodynamics within the aorta of five healthy humans were investigated to gain insight into the complex helical flow patterns that arise from the existence of asymmetries in the aortic region. The adopted approach is aimed at (1) overcoming the relative paucity of quantitative data regarding helical blood flow dynamics in the human aorta and (2) identifying common characteristics in physiological aortic flow topology, in terms of its helical content. Four-dimensional phase-contrast magnetic resonance imaging (4D PC MRI) was combined with algorithms for the calculation of advanced fluid dynamics in this study. These algorithms allowed us to obtain a 4D representation of intra-aortic flow fields and to quantify the aortic helical flow. For our purposes, helicity was used as a measure of the alignment of the velocity and the vorticity. There were two key findings of our study: (1) intra-individual analysis revealed a statistically significant difference in the helical content at different phases of systole and (2) group analysis suggested that aortic helical blood flow dynamics is an emerging behavior that is common to normal individuals. Our results also suggest that helical flow might be caused by natural optimization of fluid transport processes in the cardiovascular system, aimed at obtaining efficient perfusion. The approach here applied to assess in vivo helical blood flow could be the starting point to elucidate the role played by helicity in the generation and decay of rotating flows in the thoracic aorta.     

Morphology and ultrastructure of helical and nonhelical strains of Spiroplasma citri.

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.     

The phase transformations in starch during gelatinisation: a liquid crystalline approach

The analogy between starch and a chiral side-chain polymeric liquid crystal is examined in relation to the processes involved during gelatinisation. There are three important parameters for characterisation of the molecular phase behaviour of the amylopectin: the lamellar order parameter (psi), the orientational order parameter of the amylopectin double helices (phi), and the helicity of the sample (h, the helix/coil ratio, a measure of the helix-coil transition of the double helices). The coupling between the double helices and the backbone through the flexible spacers is affected dramatically by the water content and it is this factor which dictates the particular phase adopted by the amylopectin inside the starch granule as a function of temperature. SAXS, WAXS and 13C CP/MAS NMR are used to examine these phenomena in excess water. Furthermore, previous experimental evidence pertaining to the limiting water case is reviewed with respect to this new theoretical framework.     

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.