Studies on beta-turn of peptides. IX. Effect of 1st and 14th amino acids of tetrapeptide sequences on their beta-turn preferences studied by CD spectra of their chromophoric derivatives

The effect of changing 1st and 4th amino acid residues on beta-turn preference of tetrapeptide sequences was studied by use of CD spectra of th chromophoric derivatives, which have Dnp- and pNA-groups as the amino and carboxyl substituents, respectively. The effect was examined with the tetrapeptides having such sequences at the 2nd and 3rd positions as -L-Pro-L-Asn-, -L-Pro-Gly-, -L-Pro-D-Ala-, -L-Ala-D-Leu-, -L-Ala-L-Pro-, and -D-Ala-L-Pro-. The beta-turn preferences estimated from the CD intensities of the bands due to exciton interaction were found to depend largely on the configurations of the 1st and 4th amino acid residues. When 1st and 2nd (or 3rd and 4th) residues had the same configuration, decreased intensity of the CD band was observed even if the internal sequence had high beta-turn preference. Terminal Gly residues were favorable for the beta-turn conformation in many of the tetrapeptide sequences examined.     

Fluorescence and CD studies on the conformation of the gastrin releasing peptide in solution and in the presence of model membranes

The conformation of the heptacosapeptide hormone, gastrin releasing peptide, has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence and CD. The results obtained show that, in buffer, the hormone exists in a collection of flexible, random coil type conformers, characterized by a beta-turn between residues 14-19. On the other hand, organic solvents can induce some degree of ordered secondary structure in the peptide chain. The marked changes, observed in CD and fluorescence spectra upon addition of lysolecitin micelles and dimyristoylphosphatidylserine vesicles, clearly show that the peptide interacts with lipids, assuming a lipid specific configuration. Interestingly, no significative spectroscopic changes are produced by exposure to dimyristoylphosphatidylcholine vesicles both in the gel and liquid-chrystalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.     

Stereochemistry of peptides containing 1-aminocycloheptane-1-carboxylic acid (Ac7c).

The crystal structures of four peptides incorporating 1-aminocycloheptane-1-carboxylic acid (Ac7c) are described. Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe adopt beta-turn conformations stabilized by an intramolecular 4----1 hydrogen bond, the former folding into a type-I/III beta-turn and the latter into a type-II beta-turn. In the dipeptide esters, Boc-Aib-Ac7c-OMe and Boc-Pro-Ac7c-OMe, the Ac7c and Aib residues adopt helical conformations, while the Pro residue remains semi-extended in both the molecules of Boc-Pro-Ac7c-OMe found in the asymmetric unit. The cycloheptane ring of Ac7c residues adopts a twist-chair conformation in all the peptides studied. 1H-NMR studies in CDCl3 and (CD3)2SO and IR studies in CDCl3 suggest that Boc-Aib-Ac7c-NHMe and Boc-Pro-Ac7c-Ala-OMe maintain the beta-turn conformations in solution.     

Theoretical pi-pi absorption and circular dichroic spectra of beta-turn model peptides

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate pi-pi absorption and circular dichroic spectra of beta-turn model peptides in certain conformations. These include Ac-Gly-Gly-NHMe, Ac-L-Ala-L-Ala-NHMe, and Ac-L-Ala-Gly-NHMe in the standard beta-turn conformations I, II, and III of Venkatachalam and cyclo(L-Ala-Gly-epsilon-aminocaproyl), cyclo(L-Ala-L-Ala-epsilon-aminocaproyl), and cyclo(L-Ala-D-Ala-epsilon-aminocaproyl) in the minimum-energy conformations of Nemethy et al. Boltzmann average circular dichroic spectra of the cyclic compounds agree with experimental spectra in most respects. The results are compared with previous theoretical CD spectra for these molecules and with conformational assignments based on other evidence. Absorption spectra in the pi-pi band are predicted to be moderately sensitive to conformation.     

Ca(2+)-induced changes of surfactin conformation: a FTIR and circular dichroism study

Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.     

Tuftsin analogs and their biological activity

Summary In this paper the literature data on the structure-activity relationship for the series of tuftsin analogs are summarized. Among others, the questions of the substitution of particular amino acid residues in different positions of the peptide chain, as well as the questions of shortening and lengthening of the peptide chain of tuftsin, are reviewed. The existing models of the biologically active conformation of tuftsin are also summarized.     

Quantitative analysis of cyclic beta-turn models.

The beta-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the alpha-helix and beta-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of beta-turns. Recently, it has been reported (Hollósi, M., Kövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527-1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772-9784) that some pseudohexapeptides (e.g., the cyclo[(delta)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II beta-turns, although the X-ray-determined conformation was an ideal type I beta-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above beta-turn models has an influence on their conformational properties. Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II beta-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 beta-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major beta-turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.     

Designed beta-hairpin peptides with defined tight turn stereochemistry

The conformational analysis of two synthetic octapeptides, Boc-Leu-Val-Val-D-Pro-L-Ala-Leu-Val-Val-OMe (1) and Boc-Leu-Val-Val-D-Pro-D-Ala-Leu-Val-Val-OMe (2) has been carried out in order to investigate the effect of beta-turn stereochemistry on designed beta-hairpin structures. Five hundred megahertz (1)H NMR studies establish that both peptides 1 and 2 adopt predominantly beta-hairpin conformations in methanol solution. Specific nuclear Overhauser effects provide evidence for a type II' beta-turn conformation for the D-Pro-L-Ala segment in 1, while the NMR data suggest that the type I' D-Pro-D-Ala beta-turn conformation predominates in peptide 2. Evidence for a minor conformation in peptide 2, in slow exchange on the NMR time scale, is also presented. Interstrand registry is demonstrated in both peptides 1 and 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt beta-hairpin conformations nucleated by D-Pro-L-Ala type II' beta-turns and are stabilized by three cross-strand hydrogen bonds. CD spectra for peptides 1 and 2 show marked differences, presumably as a consequence of the superposition of spectral bands arising from both beta-turn and beta-strand conformations.     

Conformation of the RNA polymerase II C-terminal domain: circular dichroism of long and short fragments

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a beta-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix of polyproline II (P(II)) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C has P(II) and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be about 75 %, the remainder being unordered and P(II) conformations. The Tyr side-chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-aminoisobutyric acid leads to a large increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P(II) content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5-->Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P(II) and beta-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P(II) and WW or SH3 domains, or with some unknown P(II)-binding motif.     

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.     

Circular dichroism studies of some arginine-vasopressin analogues

CD spectra of arginine-vasopressin (AVP) and of its analogues substituted in position 1 and/or 7 were measured in aqueous solution at different pH values. The shapes of the CD spectra of AVP analogues substituted in position 1 are strongly influenced by the type of group attached to the beta-carbon of residue 1. The substitution of the proline residues in position 7 by N-methylalanine also leads to a change in conformation of the peptide. The differences in the CD spectra are interpreted in terms of conformational changes, which are due to the interaction of the tyrosine side chain with neighbouring residues (for 1-substituted analogues of AVP), or to that between the hexapeptide ring and acyclic tripeptide chain (for 7-substituted analogues).     

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.     

Synthesis and spectroscopy of membrane receptor proteins. The gamma subunit of the IgE receptor.

The high-affinity receptor for IgE is a tetrameric complex of subunits of the type alpha beta gamma 2. We report here conformational studies of the intact gamma subunit in trifluoroethanol and water/liposomes by circular dichroism and Fourier-transform infrared (FTIR) spectroscopy. In trifluoroethanol, the FTIR amide I' frequencies were consistent with two predominant conformational components, the beta-turn and alpha-helix, whilst in liposomes consisting of D2O and dimyristoylglycerophosphocholine (Myr2GroPCho), three components were observed. The third component present may contain some left-handed extended helix. Spectral simulation was carried out to demonstrate that the CD spectra were consistent with the component conformations identified from FTIR spectroscopy. The stimulated CD spectra were in excellent agreement with the experimental spectra. The intact gamma subunit conformation in trifluoroethanol was shown to possess 72% alpha-helical and 28% beta-turn conformations. In water/Myr2GroPCho liposomes the percentage of each conformational component present is 37%, 38% and 25% for the alpha-helix, beta-turn and extended structures, respectively. Assuming that the transmembrane fragment was alpha-helical, an excellent correlation was found between this derived alpha-helical content in water/liposomes (37%) and from hydrophobicity plots where the percentage of amino acids in the transmembrane domain is predicted by others to be 34%. It is suggested that the beta-turn detected by CD and FTIR was attributable to a 3(10) helix rather than a type I or type III reverse turn.     

Studies on beta-turn of peptides. X. Effect of the chirality of 1st and 4th amino acids of tetrapeptide sequences on their beta-turn preferences studied by conformational energy calculation

A conformational energy study was performed upon the effect of replacement of the Gly of Ac-Gly-AA2-AA3-Gly-NHCH3 by L- or D-Ala when AA2-AA3 part forms a turn conformation. When D-Ala-L-Pro constitutes the AA2-AA3 moiety, L-Ala at the 1st and 4th positions favor a beta-turn conformation of the tetrapeptide, while D-Ala residues at these positions do not. In the case of L-Pro-L-Ala at the AA2-AA3 position, the effect of replacing the two Gly residues by L- or D-Ala was shown to be just the opposite to that calculated for the D-Ala-L-Pro sequence. Terminal Gly residues are always allowable for beta-turn conformation.     

The secondary structure of echistatin from 1H-NMR, circular-dichroism and Raman spectroscopy.

Detailed biophysical studies have been carried out on echistatin, a member of the disintegrin family of small, cysteine-rich, RGD-containing proteins, isolated from the venom of the saw-scaled viper Echis carinatus. Analysis of circular-dichroism spectra indicates that, at 20 degrees C, echistatin contains no alpha-helix but contains mostly beta-turns and beta-sheet. Two isobestic points are observed as the temperature is raised, the conformational changes associated with that observed between 40 degrees C and 72 degrees C being irreversible. Raman spectra also indicate considerable beta-turn and beta-sheet (20%) structure and an absence of alpha-helical structure. Three of the four disulphide bridges are shown to be in an all-gauche conformation, while the fourth adopts a trans-gauche-gauche conformation. The 1H-NMR spectrum of echistatin has been almost fully assigned. A single conformation was observed at 27 degrees C with the four proline residues adopting only the trans conformation. A large number of backbone amide protons were found to exchange slowly, but no segments of the backbone were found to be in either alpha-helical or beta-sheet conformation. A number of turns could be characterised. An irregular beta-hairpin contains the RGD sequence in a mobile loop at its tip. Two of the four disulphide cross-links have been identified from the NMR spectra. The data presented in this paper will serve to define the structure of echistatin more closely in subsequent studies.     

Glycosylation of synthetic peptides breaks helices. Phosphorylation results in distorted structure.

Two proposed glycosylation sites are located within T cell epitopes of rabies virus glycoprotein, namely VVEDEGCTNLSGF (VF13; amino acids 29-41) and GKAYTIFNKTLM (GM12; amino acids 312-323). To explore the effects on peptide conformation due to post-translational modifications, we synthesized glycosylated and phosphorylated versions of the two peptides and compared their structures with the native peptide using CD and FT-IR spectroscopy. After the modifications, i.e., glycosylation on Asn with one or two N-acetyl-glucosamine or glucose residues or phosphorylation on Ser, the low to medium degree of helicity of the unmodified peptides disappears as indicated by CD measurements in water-trifluoroethanol mixtures. Incorporation of one sugar moiety into either peptide resulted with a high probability in a type I (III) beta-turn formation with almost identical spectra for the different peptides. Elongation of the carbohydrate in GM12 only slightly enhanced this effect. In contrast, phosphorylation of VF13 caused distorted conformation of the peptide backbone. This novel and direct demonstration of a change in secondary structure by glycosylation (or phosphorylation) might be an important element in determining peptide antigen structure and function.     

Oligopurine.oligopyrimidine tracts do not have the same conformation as analogous polypurine.polypyrimidines

The ability of tracts of synthetic oligopurine.oligopyrimidines containing both adenosine and guanosine residues to approach the conformation of analogous polypurine.polypyrimidines has been examined as a function of tract length by CD spectroscopy. Tracts of up to 19 contiguous, alternating dA and dG residues yield CD spectra that are distinctly different from that of the analogous alternating polymer. Thus the structural changes reflected in the unusual CD spectrum of poly[d(AG)].poly[d(CT)] must require even longer tract lengths. Tracts of contiguous adenosines flanked by guanosine residues were seen to approach the CD spectrum of poly[dA].poly[dT] quite slowly as a function of tract length, requiring more than 24 contiguous adenosines to give CD spectra similar to the homopolymer. These results lead us to the conclusion that oligopurine tracts in vivo are not well modeled by synthetic polypurine.polypyrimidines with one or two base pair repeating units.     

CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix.

Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity.     

Chemical glycosylation of peptide T at natural and artificial glycosylation sites stabilizes or rearranges the dominant reverse turn structure.

Peptide T (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH), a fragment of HIV gp120, has been reported to inhibit binding of the virus to the CD4 receptor. The peptide assumes a beta-turn secondary structure, and stabilization of the conformation may increase the biological activity. We synthesized the octapeptide and its C-terminal pentapeptide fragment, unmodified and glycosylated, when monosaccharides were walked through the molecules. Incorporation of the sugar into the longer peptide resulted in the stabilization of the type I (III) beta-turn, as indicated by circular dichroism measurements. While N-terminal glycosylation of the shorter peptide also stabilized the type I (III) beta-turn, the circular dichroism spectra revealed slightly different type II beta-turn structures when the carbohydrate moiety was incorporated into mid-chain or C-terminal positions. Modification of biologically active reverse-turn structures by glycosylation offers a viable alternative to the peptide mimetics approach in drug design.     

Combined effect of the DeltaPhe or DeltaAla residue and the p-nitroanilide group on a didehydropeptides conformation

Two series of dehydropeptides of the general formulae Boc-Gly-X-Phe-p-NA, Boc-Gly-Gly-X-Phe-p-NA, Gly-X-Gly-Phe-p-NA.TFA, and Boc-Gly-X-Gly-Phe-p-NA, with X = Delta(Z)Phe and DeltaAla, were studied with NMR in DMSO and CDCl(3)-DMSO, and with CD in MeOH, MeCN, and TFE. The NMR spectra measured in DMSO suggest that peptides with the DeltaPhe residue next to Phe are folded whereas peptides with Gly between DeltaPhe and Phe are less ordered. NMR spectra of DeltaAla-containing peptides indicate that these peptides are flexible and their conformational equilibria are populated by many different conformations. The CD spectra show that conformational properties of the peptides studied are distinctly influenced by a mutual position of the dehydroamino acid residue and the p-NA group. They indicate that all dehydropeptides with the DeltaPhe residue, Boc-Gly-DeltaAla-Phe-p-NA, and Boc-Gly-Gly-DeltaAla-Phe-p-NA adopt ordered conformations in all solvents studied, presumably of the beta-turn type. The last two peptides exhibit surprising chiroptical properties. Their spectra show exciton coupling-like couplets in the region of the p-NA group absorption. This shape of CD spectra suggests a rigid, chiral conformation with a fixed disposition of the p-NA group. The CD spectra indicate that Boc-Gly-DeltaAla-Gly-Phe-p-NA and Gly-DeltaAla-Gly-Phe-p-NA.TFA are unordered, independently of the solvent.