Conformation of cyclobradykinin by nmr and distance geometry calculations

The conformation of the head-to-tail cyclic analogue of bradykinin in DMSO was investigated by nmr. Three sets of resonances were detected and fully assigned. These were attributed to the presence of three stable conformers, two of which were exchanging on the nmr time scale. A fourth, incomplete set of resonances was detected but not assigned. The three major conformers differ in the conformation at the three X-Pro bonds present. From nuclear Overhauser effect spectroscopy (NOESY) spectra, three sets of interproton distances were derived and used in NOE-restrained distance geometry calculations. The resulting structures were refined by energy minimization to yield families of structures. Conformer I is characterized by the presence of two type VIb β-turns between Arg¹ and Gly⁴ and between Phe⁵ and Phe⁸. The first β-turn is present also in conformer II, while an inverse γ-turn bridging Pro³ is the most pronounced structural feature of conformer III. © 1995 John Wiley & Sons, Inc.     

1H-NMR analysis of CD3-ϵ reveals the presence of turn-helix structures around the ITAM motif in an otherwise random coil cytoplasmic tail

The conformation adopted in solution by the cytoplasmic tail of CD3-ϵ has been analyzed by ¹H-nmr. The cytoplasmic tail is mostly random coil except for the amino acids conforming the immunoreceptor tyrosine-based activation motif (ITAM), YxxL/IxxxxxxxY xxL. Although the N-terminal Y xxL sequence of the motif is poorly folded, adopting 6-residue turn-like conformations with the Tyr side chain in two different orientations, the C-terminal Y xxL sequence is placed in a more complex structure involving a set of nonclassical α-helix turns and β-turns that comprises 11 amino acids. This structure is not modified by phosphorylation of the tyrosine residue. The differences in the conformation adopted around the two tyrosines of the ITAM motif suggest that they may play different roles pertaining to either binding signal transducing proteins or, alternatively, proteins involved in other processes such as endoplasmic reticulum location. © 1997 John Wiley & Sons, Inc. Biopoly 42: 75–88, 1997     

Conformation-function relationships in hydrophobic peptides: interior turns and signal sequences

Two studies are diescribed in which synthetic peptides have been designed and examined to address biochemical problems inherent in hydorphobic environments: (1) The cyclic hexapeptide cyclo-(D -Tyr(Bzl)-Gly-Ile-Leu-Gln-Pro) was synthesized as a model of an interior β-turn from the protein lysozyme. Conformational analysis by proton nmr methods, including two-dimensional nulcear Overhauser effect spectroscopy, revealed that the model peptide adopts one conformation in chloroform/dimethyl sulfoxide (98.2) and tetramethylene sulfone solutions. The conformation consists of two linked β-turns, one with the same sequence (Gly-Ile-Leu-Gln) and geometry (Type I) as the protein turn. (2) Major portions of the λ-receptor protein (LamB) signal sequences from E. coli wildtype and mutant strains have been synthesized. The conformational properties and membrane interactions of these synthetic signal peptides correlate with the in vivo export function of the wild type and mutant strains. Functional signal sequences are significantly richer in α-helix in aaqueous trifluoroethanol, lysolecithin, or sodium do-decyl sulfate solution than is a nonfunctional mutant signal sequence.     

Effect of Stereochemistry (Z and E) and Position of α,β-Dehydrophenylalanine (ΔPhe) on β-turn Stability

Several model peptides containing α, β-dehydrophenylalanine (ΔPhe) in both Z and E configurations were studied for β-turn stability at the AM1 level of theory. Both configurations of ΔPhe are well able to stabilize β-turns in the backbone. However, the β-turns for peptides bearing Z-ΔPhe are energetically more stable than the E-counterparts. The difference in energies between the global minima of these peptides having the Z and E configuration of ΔPhe, is dictated by the size and stereochemistry of residues flanking ΔPhe. One distinct feature of E-ΔPhe is that it pushes peptides to adopt a Type II β-turn with the ΔPhe residue in the (i + 1) position of the turn. This unique feature may be exploited in peptide design.     

Modified chemotactic peptides: Synthesis,conformation, and activity of HCO-Thp-Ac6c-Phe-OMe

HCO-Thp-Ac₆c-Phe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent HCO-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 contains 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 1-aminocyclohexane-1-carboxylic acid (Ac₆c) as achiral, conformationally restricted mimics of Met and Leu, respectively. In the crystal, the formyltripeptide adopts an helical conformation at the Thp and Ac₆c residues, of the type α_R and α_L, respectively, whereas the C-terminal phenylalanine is quasi-extended. A system of two consecutive γ-turns, centered at the first two residues, better explains the nmr data as compared with an alternative β-turn structure. The conformation of the new analogue 3 is compared with those of two related peptides containing Thp as N-terminal residue. The biological activity of 3 has been determined on human neutrophils and compared to that of the previously studied model [Ac₆c²] fMLP-OMe. While the above analogue is highly active in the superoxide anion production, the new tripeptide 3 is practically unable to elicit any of the tested biological activities. © 1996 John Wiley & Sons, Inc.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

On the location of β-turns

β-Turns are a common feature of cyclic peptides, but judging from recent x-ray and solution studies of cyclic hexapeptides it is not always possible to predict in advance the type of turn and the position of the turns in the sequence. Two or more backbone conformations containing β-turns may be of comparable energy and in rapid solvent- and temperature-dependent equilibrium in solution. The use of differential relaxation effects produced by a nitroxyl radical to locate β-turns with only minor perturbation of such equilibria is noted. Examination of the effect of a nitroxyl on the N-H resonances of the decapeptide hormone luteinizing hormone releasing hormone supports a dominant conformation with a β-turn at Gly⁶-Leu⁷. Although this turn is probably part of the biologically active conformation, it is not obvious in the more active [D -Ala⁶] analog.     

Chain length effects on helix-hairpin distribution in short peptides with Aib-DAla and Aib-Aib segments

The Aib-D Ala dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and Boc-Aib-Xxx-NHMe (4, 8), where Xxx=DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7), and Boc-Aib-Aib-NHMe (8) helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3 OH. The β-turn conformations (type-I'/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc-Aib-D Ala-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 3(10)-helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc-Val-Aib-DAla-Leu-NHMe (3) adopts a novel α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of an NOE between Val (1) NH?HNCH3 (5) in CD3OH suggests, that the solid state conformation is maintained in methanol solutions.     

Lecithin cholesterol acyltransferase (LCAT) activity in the presence of Apo-AI-derived peptides exposed to disorder–order conformational transitions

Although the association of Apo AI with HDLs has been proposed to activate LCAT activity, the detailed molecular mechanisms involved in the process are not known. Therefore, in this study we have investigated how conformational changes in several exposed regions of Apo-AI might cause LCAT activation and for this purpose, designed a strategy to investigate three Apo AI-derived peptides. Since these peptides present the ability to adopt several secondary structure conformations, they were used to determine whether LCAT activity could be modulated in the presence of a particular conformation. Circular dichroism experiments showed that Apo AI-derived peptides in PBS displayed a disordered arrangement, with a strong tendency to adopt β-sheet and random conformational structures as a function of concentration. However, in the presence of Lyso-C₁₂PC, maximal percentages of α-helical structures were observed. Performed in human plasma, time-course experiments of LCAT activity under control conditions reached the highest level of ³H-cholesteryl esters after 2.5 h incubation. In the presence of Apo AI-derived peptides, a significant increase in the production of ³H-cholesteryl esters was observed. The present study provides an important insight into the potential interactions between LCAT and lipoproteins and also suggests that peptides, initially present in a disordered conformation, are able to sense the lipid environment provided by lipoproteins of plasma and following a disorder-to-order transition, change their conformation to an ordered α-helix.     

Multiple different missense mutations in the pore region of HERG in patients with long QT syndrome

Long QT syndrome (LQTS), is an inherited cardiac disorder in which ventricular tachyarrhythmias predispose affected individuals to syncope, seizures, and sudden death. Characteristic electrocardiographic findings include a prolonged QT interval, T wave alternans, and notched T waves. We have screened LQTS patients from 89 families for mutations in the pore region of HERG , the K⁺ channel gene previously associated with chromosome 7-linked LQT2. In six unrelated LQTS kindreds, single-strand conformation polymorphism analyses identified aberrant conformers in all affected family members. These conformers were not seen in over 100 unaffected, unrelated control individuals, suggesting that they represent pathogenic LQTS mutations. DNA sequence analyses of the aberrant conformers demonstrated that they reflect five different missense mutations: V612L, A614V, N629D, N629S, and N633S. The missense mutation A614V was found in two unrelated families. Further functional studies will be required to determine what effect each of these changes may have on HERG channel function. Received: 15 July 1997 / Accepted: 10 November 1997     

Conformational difference between diastereomers of Dnp-Val-Aib-Gly-Leu-pNA studied by x-ray crystal analyses

In order to investigate the Conformational change of the α-aminoisobutyric acid (Aib) containing peptide by the D /L replacement of an amino acid residue, single crystals of two diastereomers, Dnp-L -Val-Aib-Gly-L -Leu-pNA (L -L isomer) and Dnp-D -Val-Aib-Gly-L -Leu-pNA (D -L isomer), were prepared from aqueous methanol solutions as CH₃OH and CH₃OH · H₂O solvates, respectively, and were analyzed by the x-ray diffraction method. Molecular conformation of L -L isomer adopts consecutive two different types of β-turns, a type II′ β-turn bent at Aib-Gly, and a type III β-turn bent at Gly-Leu, stabilized by two intramolecular (Leu) NH …? O?C (Val) and (pNA) NH …? O?C(Aib) hydrogen bonds. In contrast, these two intramolecular hydrogen bonds lead the D -L isomer to a distorted 3₁₀-helix conformation consisting of consecutive two type-III β-turn of Aib-Gly-Leu sequence. The most significant structural difference between these diastereomers is the mutual orientation between the Dnp and pNA chromophores. While the extensive stacking of both the chromophores is intramolecularly formed for the folded conformation of L -L isomer, they are oriented toward an opposite direction in the open conformation of D -L isomer and are intermolecularly stacked with each other. The large separation between these diastereomers observed in the chromatography is discussed in the relation with their Conformational differences. © 1993 John Wiley & Sons, Inc.     

Solution structure of regioselectively addressable functionalized templates: An NMR and restrained molecular dynamics investigation

Three cyclic peptides that are Regioselectively Addressable Functionalized Templates (RAFT) for use in protein de novo design have been investigated using a combination of nmr, restrained molecular dynamics, and CD spectroscopy. These peptides contain up to four selectively addressable sites (orthogonally protected lysine side chains) or have selectively addressable faces. The results show a common stable conformation for templates of this kind based on two type II β-turns and an associated β-sheet structure. A preferential orientation for the side chains is also demonstrated. The significance of these findings is discussed in the context of applications of RAFT that rely on their conformational rigidity and ability to present functionalities in a defined spatial arrangement. © 1996 John Wiley & Sons, Inc.     

Order-disorder conformational transitions of the hydrocarbon chains of lipids

In many lipid-containing systems (intact membranes, lipid-water and proteinlipid-water phases) the hydrocarbon chains are known to undergo a reversible temperature-dependent transition between a highly disordered (type α) and a partly ordered (type β) conformation; in the β conformation the chains, stiff and all parallel, are packed with rotational disorder according to a two-dimensional hexagonal lattice. This work describes an X-ray diffraction and freeze-fracturing electron microscope study of the phases involved in this conformational transition. Several lipid-water systems were studied: mitochondrial lipids; phosphatidic acid, synthetic lecithin; hen egg lecithin. The conformational transition is found to be a complex phenomenon dependent upon the chemical composition of the lipids, the amount of water and temperature. When the lipid is a pure chemical species the transition involves two phases; one with all the chains in the α conformation the other with all the chains in the β conformation. If the chains are heterogeneous, then from the onset of the transition from type α, they segregate into regions with different conformation, presumably according to their length and degree of saturation. One of the phases (Lαβ) consists of regularly stacked lipid lamellae, each of which is a disordered mosaic of two types of domains; one with the chains in the α, the other in the β conformation. In another phase (Lγ) each lipid lamella is formed by one monolayer of type α and one of type β, joined by their apolar faces. Two other phases (Pγ and Pαβ) display two-dimensional lattices, and consist of lipid lamellae distorted by wave-like ripples, with an ordered segregation of domains in the α and in the β conformation. The number and the structure of the phases involved in the conformational transition are strongly dependent upon the heterogeneity of the hydrocarbon chains and upon the charge and hydration of the polar groups. The results of this study have a bearing on the conformation of the chains in membranes, and on the possible biological significance of conformational transitions.     

Structure and Dynamics of Elastin Building Blocks. Boc-LG-OEt,Boc-VG-OEt,Boc-VGG-OH

Abstract

Short di- and tripeptides such as Boc-LG-OEt, Boc-VG-OEt and Boc-VGG-OH, corresponding to abundant repetitive sequences in elastin, have been extensively studied both in solid state, by X-ray diffraction, and in solution by circular dicroism and nuclear magnetic resonance. Furthermore, theoretical procedures such as simulated annealing and molecular dynamics were also performed on these peptides.

In general, the results indicate that no one single structure (be folded or extended) could be representative for these sequences in the protein, but rather that a multiplicity of interconverting conformers, ranging from folded to extended structures, should be considered. In any case, these structures, e.g. β-turns, polyglycine II and β-conformations, are those previously suggested to participate to conformational equilibria of elastin.     

Insulin analogues with modifications in the β-turn of the B-chain

The β-turn formed by the amino acid residues 20–23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of β-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the β-turn. In two analogues, we have substituted α-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potencyca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the β-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.     

Structural features determining the site specificity of a rat liver cAMP-independent protein kinase.

The Seryl and Threonyl residues affected in α_s1 and in β-caseins by rat liver “casein kinase TS” (a cytosolic cAMP-independent protein kinase) have been identified. All of them, as well as the residues affected by the same enzyme in α_s2-casein are characterized by an acidic group two residues to their C terminus and by being located within predicted β-turns. Several other potential sites of phosphorylation, according to their primary structure, but located outside predicted β-turns, are not significantly labeled by the protein kinase. It seems conceivable therefore that both a definite aminoacid sequence including a critical acidic residue, and the existence of a β-turn are required for the activity of this protein kinase.     

Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol-water mixtures: Analysis of CD spectra

The cytolytic activities and conformational properties of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE), a 33-residue linear peptide that exhibits unusual shark repellent and cytolytic activities, and its analogues have been examined in aqueous environment and trifluoroethanol (TFE) using CD spectroscopy. A peptide corresponding to the 1–26 segment and an analogue where P7 has been changed to A show greater hemolytic activity than pardaxin. While the peptide corresponding to the N-terminal 18-residue segment does not exhibit hemolytic activity, its analogue where P7 is replaced by A is hemolytic. The secondary structural propensities of the peptides were inferred by deconvolution of the experimental spectra into pure components. Pardaxin, its variant where proline at position 7 was replaced by alanine, and shorter peptides corresponding to N-terminal segments exist in multiple conformations in aqueous medium that are comprised of β-turn, β-sheet, and distorted helical structures. With increasing proportions of TFE, while helical conformation predominates in all the peptides, both distorted and the regular α-helices appear to be populated. Analysis of CD spectra by deconvolution methods appears to be a powerful tool for delineating multiple conformations in peptides, especially membrane-active peptides that encounter media of different polarity ranging from aqueous environment to one of low dielectric constant in the hydrophobic interior of membranes. Our study provides further insights into the structural requirements for the biological activity of pardaxin and related peptides. © 1997 John Wiley & Sons, Inc. Biopoly 41: 635–645, 1997     

Prediction of β-sheets in immunoglobulin chains. Comparison of various methods and an expanded 20 × 20 table for evaluation of the effects of nearest-neighbors on conformations of middle amino acids in proteins

The extraordinarily large number of immunoglobulins renders them an intriguing class of molecules for attempts to predict their conformations. The predictive method applied, using a 20 × 20 table of the observed effects of nearest-neighboring amino acids on the conformation (Φ,Ψ angles) of the middle residue in known proteins, indicates positions of tri-peptides that tend to break α-helices or regular β-sheets. This 20 × 20 table is derived from data on 19 proteins, as compared with the earlier version based on 12 proteins, and includes a separate listing of residues of β-turns that have helical Φ,Ψ values. Secondary conformations predicted by methods of Chou and Fasman, Lim and Burgess, Ponnuswamy, and Scheraga have also been compared; for all three methods, wrong predicitons of residues in β-sheet conformation exceed correct ones. Better predictions are obtained when there is agreement with two or three of the methods. If there is consistent overprediction of β-structure, as with the Chou and Fasman method, the use of the β-sheet-breaking tripeptides can improve pre-dictability somewhat.     

Structural aspects of Ca2+ binding by acyclic peptides: low-energy conformational domains and molecular dynamics of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide.

We have applied random-search, energy minimization and molecular dynamics simulations to investigate the structural aspects of the interaction of N-acetyl-L-prolyl-D-alanyl-L-alanine-N'-methylamide with Ca2+. Spectral data on related peptides had suggested that the beta-turn conformation might be a prerequisite for the binding of cation ion by such short linear peptides. In order to relate the conformational characteristics with the Ca(2+)-binding affinities of these peptides, the molecular events involved in cation binding need to be understood. We have addressed this problem in this study by using a systematic approach that involved the following steps. First, a random search technique was used to generate a large population of conformers for the free peptide in the absence of Ca2+. Next, the energies of these conformers were computed. Conformations with energies within 4 kcal/mol of the global minimum were analysed and found to fall into four main groups characterized by the presence of different types of hydrogen-bonded structures including single and consecutive beta-turns. The energies for interconversion of conformers from one group to another were computed and found to be relatively small (< 10 kcal/mol). Finally, molecular dynamics of the peptide at 300K in the presence of Ca2+ were used to simulate the cation binding process. Starting points for these simulations were generated by placing the ion in the vicinity of two molecules of the peptide. The simulation results showed that the conformers with two consecutive beta-turns led to the formation of a stable 2:1 (peptide:Ca2+) sandwich complex in agreement with earlier experimental observations on similar linear peptides. While the starting conformation of the peptide in the consecutive beta-turn structure allowed for the proper orientation of three carbonyl oxygen atoms for chelation to the metal ion, the dynamics of complex formation rearranged the peptide structure substantially, leading to the formation of an 8-coordinated Ca2+ complex in a dodecahedral spatial arrangement. Thus, based on the energetics of the structures and processes involved, the present study demonstrates that: a) peptide-Ca2+ complex formation is initiated by conformers adopting consecutive beta-turn structures which subsequently go over to a significantly different conformation found in the complex; and, b) The facile interconversion between the low-energy conformers in the different groups would help shift the equilibrium population towards the consecutive beta-turn structure during the complex formation.     

Alternatingly twisted β-hairpins and nonglycine residues in the disallowed II′ region of the Ramachandran plot

The structure of the SH3 domain of α-spectrin (PDB code 1SHG) features Asn47 in the II′ area of the Ramachandran plot, which as a rule admits only glycine residues, and this phenomenon still awaits its explanation. Here, we undertook a computational study of this particular case by means of molecular dynamics and bioinformatics approaches. We found that the region of the SH3 domain in the vicinity of Asn47 remains relatively stable during denaturing molecular dynamics simulations of the entire domain and of its parts. This increased stability may be connected with the dynamic hydrogen bonding that is susceptible to targeted in silico mutations of Arg49. Bioinformatics analysis indicated that Asn47 is in the β-turn of a distinctive structural fragment we called ‘alternatingly twisted β-hairpin.’ Fragments of similar conformation are quite abundant in a nonredundant set of PDB chains and are distinguished from ordinary β-hairpins by some surplus of glycine in their β-turns, lack of certain interpeptide hydrogen bonds, and an increased chirality index. Thus, the disallowed conformation of residues other than glycine is realized in the β-turns of alternatingly twisted β-hairpins.