Structuro-functional organization of delta sleep-inducing peptide

Theoretical conformational analysis was carried out for a nonapeptide hormone (delta sleep-inducing peptide). Possible structure of the neuropeptide under physiological conditions may be described by a set of low-energy conformations belonging to nine different forms of the backbone. A solution of the "reverse conformational problem" for delta sleep inducing peptide enables one to predict modified amino acid sequences (D-Ala3-, Pro4-, Pro6-, Pro7, and Tyr7-analogs), which may assume one of the low-energy states of the native hormone. The influence of the solute was not taken into account in our calculations.     

Spatial structure of BAM-12P dodecapeptide and its analogues

Theoretical conformational analysis was used to study the spatial structure and conformational properties of the bovine adrenal medulla dodecapeptide BAM-12P (Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12). Twenty-three low-energy conformations of the BAM-12P backbone were shown to represent the spatial structure of the peptide. The inverse structural problem was solved, and synthetic analogues of BAM-12P were proposed, the spatial structures of which correspond to a set of low-energy potentially physiologically active conformations of the natural dodecapeptide. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Spatial Structure of BAM-12P Dodecapeptide and Its Analogues

Theoretical conformational analysis was used to study the spatial structure and conformational properties of the bovine adrenal medulla dodecapeptide BAM-12P (Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12). Twenty-three low-energy conformations of the BAM-12P backbone were shown to represent the spatial structure of the peptide. The inverse structural problem was solved, and synthetic analogues of BAM-12P were proposed, the spatial structures of which correspond to a set of low-energy potentially physiologically active conformations of the natural dodecapeptide.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 245–250.Original Russian Text Copyright © 2005 by Akhmedov, Tagiev, Hasanov, Makhmudova.     

Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.     

Studies on the conformation and interactions of elastin: nuclear magnetic resonance of the polyhexapeptide.

Synthesis, proton magnetic resonance and carbon-13 magnetic resonance characterizations, including complete assignments, are reported for the polyhexapeptide of elastin, HCO-Val(Ala1-Pro2-Gly3-Val4-Gly5-Val6)18-OMe. Temperature dependence of peptide NH chemical shifts and solvent dependence of peptide C-O chemical shifts have been determined in several solvents and have been interpreted in terms of four hydrogen bonded rings for each repeat of the polyhexapeptide. The more stable hydrogen bonded ring is a beta-turn involving Ala1C-O--HN-Val4. More dynamic hydrogen bonds are an 11-atom hydrogen bonded ring Gly3NH--O-C Gly5, a 7-atom hydrogen bonded ring (a gamma-turn) Gly3 C-O--NH-Gly5, and a 23-atom hydrogen bonded ring Val6inH--O-C Val6(i+1). This set of hydrogen bonds results in a right-handed beta-spiral structure with slightly more than two repeats (approximately 2.2) per turn of spiral. The beta-spiral structure is briefly discussed relative to data on the elastic fiber.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.     

D-Ala5 analog of the elastin polypentapeptide. Physical characterization

The D-Ala5 analog, (L-Val1-L X Pro2-Gly3-L X Val4-D-Ala5) of the polypentapeptide (PPP) of elastin is synthesized and characterized by a series of physical methods. Carbon-13 and proton nuclear magnetic resonance spectroscopies are used to verify purity and, by means of solvent dependence of peptide C-O chemical shift and of temperature dependence of peptide NH chemical shift, to establish by comparison with the PPP of elastin the presence and increased stability of the Type II Pro2-Gly3 beta-turn. The temperature dependence of aggregation in water to form a viscoelastic phase called the coacervate is reported for several concentrations. Comparison of carbon-13 nuclear magnetic resonance spectra obtained under identical conditions for the coacervate states of the PPP of elastin and the D-Ala5 analog shows the effect of replacing the Gly5 residue by a D-Ala5 residue to be one of greatly restricting mobility of the polypeptide chain. Scanning electron micrographs, of the coacervate alone and of the coacervate cross-linked and compounded to a Dacron fabric before and after stress-strain studies, are reported which show the D-Ala5 PPP matrix to rupture during the stresses of drying and of stretching while wet. Thus, the effect of adding a methyl moiety to the Gly5 residue of the PPP of elastin is to decrease markedly the mobility of the polypeptide chain and to destroy elasticity. The results are presented as a test of the proposed librational entropy mechanism of elasticity of the PPP of elastin.     

Designing of peptides with left handed helical structure by incorporating the unusual amino acids

The conformational behaviour of delta Ala has been investigated by quantum mechanical method PCILO in the model dipeptide Ac-delta Ala-NHMe and in the model tripeptides Ac-X-delta Ala-NHMe with X = Gly, Ala, Val, Leu, Abu and Phe and is found to be quite different. The computational results suggest that in the model tripeptides the most stable conformation corresponds to phi 1 = -30 degrees, psi 1 = 120 degrees and phi 2 = psi 2 = 30 degrees in which the > C = 0 of the acetyl group is involved in hydrogen bond formation with N-H of the amide group. Similar results were obtained for the conformational behaviour of D-Ala in Ac-D-Ala-NHMe and Ac-Ala-D-Ala-NHMe. The conformational behaviour of the amino acids delta Ala, D-Ala, Val and Aib in model tripeptides have been utilized in the designing of left handed helical peptides. It is shown that the peptide HCO-(Ala-D-Ala)3-NHMe can adopt both left and right handed helix whereas in the peptide Ac-(Ala-delta Ala)3-NHMe the lowest energy conformer is beta-bend ribbon structure. Left handed helical structure with phi = 30 degrees, psi = 60 degrees for D-Ala residues and phi = psi = 30 degrees for delta Ala is found to be more stable by 4 kcal mole-1 than the corresponding right handed helical structure for the peptide Ac-(D-Ala-delta Ala)3-NHMe. In both the peptides Ac-(Val-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe the most stable conformer is the left handed helix. Comparisons of results for Ac-(Ala-delta Ala)3-NHMe and Ac(Val-delta Ala)3-NHMe and Ac-(D-Ala-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe also reveal that the Val residues facilitate the population of 3(10) left handed helix over the other conformers. It is also shown that the conformational behaviour of Aib residue depends on the chirality of neighbouring amino acids, i.e. Ac-(Aib-Ala)3-NHMe adopts right handed helical structure whereas Ac-(Aib-D-Ala)3-NHMe is found to be in left handed helical structure.     

Conformation and calcium-binding properties of a bicyclic nonapeptide

The conformation and calcium binding properties of the bicyclic nonapeptide BCP2, cyclo-(Glu(1)-Ala(2)-Pro(3)-Gly(4)-Lys(5)-Ala(6)-Pro(7)-Gly(8))-cyclo-(1gamma --> 5epsilon) Gly(9), have been investigated by means of NMR spectroscopy. Interproton distances, evaluated by nuclear Overhauser effect (NOE) contacts, and straight phi angles, from (3)J(NH-alphaCH), have been used to obtain a feasible model for the BCP2-Ca(2+) (BCP: bicyclic peptide) complex by means of restrained molecular dynamics (RMD). The NMR analysis of the free peptide, carried out in CD(3)CN, shows the presence in solution of at least four conformers in intermediate exchange rate. The addition of calcium ions caused the appearance of a new set of resonances, differing from those observed for the free BCP2. A comparison with published data about the conformational behavior of the closely analogous peptide BCP3, differing from BCP2 for two Leu residues instead of two Ala residues in positions 2 and 6, shows that this simple substitution dramatically increases the peptide flexibility. On the contrary, upon calcium ion addition, both BCP2 and BCP3 reach a strictly close conformation, as strongly testified by the almost identical (1)H-NMR spectra exhibited by both peptides. The RMD molecular model of the BCP2-Ca(2+) complex, here reported, is a quite symmetric structure, presenting a three-dimensional cavity ideal for the binding of spherical cations. Four carbonyls from the main ring (Ala(2), Gly(4), Ala(6) and Gly(8)) point toward it, offering, together with the two carbonyls of the peptide bridge (Gly(9) and gammaGlu(1)), putative coordinations to the cation.     

Structure-functional organization of the secretin molecule. II. Direct conformation problem

Spatial structure of peptide hormone secretin was investigated by the theoretical conformational method. A solution of the "direct conformational problem" for this hormone indicated that the possible structure of the secretin molecule under polar conditions may be described only by two families of low-energy conformations, possessing relatively conformational valid (Thr7-Leu22) and variable (His1-Phe6 and Leu23-Val27NH2) fragments. One of these families is comprised by five twists of the alpha-helix, while the second isoenergetic family possesses two short segments of the alpha-helix, divided by an irregular structure of the tetrapeptide.     

NMR study of a lymphocyte differentiating thymic factor. An investigation of the Zn(II)-nonapeptide complexes (thymulin)

Detailed investigations of a serum peptide (less than Glu1-Ala2-Lys3-Ser4-Gln5-Gly6-Gly7-Ser8-++ +Asn9) were carried out by 1H and 13C NMR spectroscopy to elucidate the structure of the complex formed with Zn(II), thymulin, which has been found to be active in vivo. These experiments were performed in dimethyl sulfoxide-d6 solution at different metal:peptide ratios. The results suggest the following conclusions. (i) The Zn(II) complexation corresponds to a fast exchange on the NMR time scale. (ii) The evolution of 1H and 13C NMR chemical shifts indicates the existence of two types of complexes: a 1:2 species associating two peptide molecules and one Zn(II) ion and a complex with 1:1 stoichiometry. The former is predominant for metal:peptide ratios below unity. (iii) In the 1:2 complex, Zn(II) is coordinated by the Ser4-O gamma H and Asn9-CO2- sites, while in the 1:1 complex, Ser8-O gamma H is the third ligand to the Zn(II) ion. The results are compared with those for the [Ala4] and [Ala8] analogues, and those for the complexes of thymulin with other metal ions (Cu2+ and Al3+) in terms of its biological activity. These comparative studies suggested that the 1:1 complex is the only conformation recognized by the antibodies.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly].

A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.     

NMR constrained solution structures for laminin peptide 11. Analogs define structural requirements for inhibition of tumor cell invasion of basement membrane matrix.

Peptide 11, CDPGYIGSR-NH2, is a segment of laminin which blocks tumor cell invasion. A high affinity laminin receptor in tumor cells is thought to be blocked by the carboxyl-terminal YIGSR, and conformational energy calculations suggest that the glycine in YIGSR allows an important conformational bend. We replaced the YIGSR glycine residue in peptide 11 with either D-alanine or L-alanine to allow or disfavor the proposed glycine bend. We found the Gly7-->D-Ala7 analog to be equal to peptide 11 in inhibiting tumor cell invasion of basement membrane matrix. The Gly7-->L-Ala7 analog was much less capable of invasion inhibition. Two-dimensional 1H-1H NMR was used to study the solution conformations of the peptide 11 analogs. NOESY experiments revealed close NH-NH contacts in peptide 11 and the D-Ala7 analog, but not in the L-Ala7 analog. Molecular dynamics generated low energy structures with excellent NOE agreement for peptide 11 and its analogs. Both peptide 11 and the D-Ala7 analog, but not the less active L-Ala7 analog, were predicted to have similar bends around Gly7 or D-Ala7. These results suggest that a bend in the YIGSR region of peptide 11 may be important for the binding of laminin to its metastasis-associated receptor.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

Conformation of the eosinophil chemotactic tetrapeptides and analogues in dimethyl sulfoxide. 1H n.m.r. studies

Proton nuclear magnetic resonance parameters are reported for DMSO-d6 solutions of the eosinophil chemotactic tetrapeptides, Val1-Gly2-Ser3-Glu4 and Ala1-Gly2-Ser3-Glu4, as well as three analogues of the Val1 tetrapeptide, D-Val1, Ala2 and Ala3. The synthesis of Val-(S)-[alpha-2 H1] Gly-Ala-Glu, in which the glycine has been stereospecifically deuterated in the H alpha 3 position, has allowed the assignment of the 1H resonances belonging to individual H alpha 2 and H alpha 3 glycine methylene protons. Simulation of the glycine ABX spin system yields two vicinal coupling constants which are consistent with a highly preferred conformation about the glycine HN-C alpha bond. The chemical shifts, coupling constants, temperature coefficients of amide proton chemical shifts and calculated side chain rotamer populations are reported for all peptides. The coupling constant analysis and temperature coefficients of amide proton chemical shifts together suggest that a type I beta-turn conformation is preferred by the Ala3 analogue. The 1H n.m.r. parameters of the other peptides suggest that these can also adopt a beta-turn conformation in DMSO. There are, however, considerable differences in the extent of conformational averaging undergone by the various peptides.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.     

The occurrence of different kappa opioid receptors (K1 and K2) in frog (Rana esculenta) brain membranes

Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.