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.     

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.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Increasing protein conformational stability by optimizing beta-turn sequence

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Reactivity toward deamidation of asparagine residues in beta-turn structures.

Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn.     

Analysis and prediction of the different types of beta-turn in proteins

beta-Turns have been extracted from 59 non-identical proteins (resolution 2 A) using the standard criterion that the distance between C alpha (i) and C alpha (i + 3) is less than 7 A (1 A = 0.1 nm). The beta-turns have been classified, using phi, psi angles, into seven conventional turn types (I, I', II, II', IV, VIa, VIb) and a new class of beta-turn, designated type VIII, in which the central residues (i + 1, i + 2) adopt an alpha R beta conformation. Most beta-turn types are found in various topological environments, with the exception of I' and II' beta-turns, where 83% and 50%, respectively, are found in beta-hairpins. Sufficient data have been gathered to enable, for the first time, the separate statistical analysis of type I and II beta-turns. The two turn types have been shown to be strikingly different in their sequence preferences. Type I turns favour Asp, Asn, Ser and Cys at i; Asp, Ser, Thr and Pro at i + 1; Asp, Ser, Asn and Arg at i + 2; Gly, Trp and Met at i + 3, whilst type II turns prefer Pro at i + 1; Gly and Asn at i + 2; Gln and Arg at i + 3. These preferences have been explained by the specific side-chain interactions observed within the X-ray structures. The positional trends for type I and II beta-turns have been incorporated into the simple empirical predictive algorithm originally developed by P.N. Lewis et al. The program has improved the positional prediction of beta-turns, and has enhanced and extended the method by predicting the type of beta-turn. Since the observed preferences reflect local interactions these predictions are applicable not only to proteins, but also to peptides, many of which are thought to contain beta-turns.     

Conformation-activity relationship of tachykinin neurokinin A (4-10) and of some [Xaa8] analogues.

NKA (4-10), the C-terminal heptapeptide fragment (Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) of tachykinin NKA, is more active than the parent native compound in the interaction with the NK-2 receptor. Substitution of Gly8 with the more flexible residue beta-Ala8 increases its selectivity with respect to other two known receptors (NK-1 and NK-3), whereas substitution with either D-Ala8 or GABA8 deprives the peptide of its biological activity. These findings can be interpreted by a conformational analysis based on NMR studies in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture combined with internal energy calculations. NKA(4-10) is characterized by a structure containing a type I beta-turn extending from Ser5 to Gly8, followed by a gamma-turn centered on Gly8, whereas for [beta-Ala8]NKA(4-10) is possible to suggest a type I beta-turn extending from Ser5 to beta-Ala8, followed by a C8 turn comprising beta-Ala8 and Leu9 and by another beta-turn extending from beta-Ala8 to the terminal NH2. The preferred conformation of [beta-Ala8]NKA(4-10) is not compatible with models for NK-1 and NK-3 agonists proposed on the basis of rigid peptide agonists [Levian-Teitelbaum et al. (1989) Biopolymers 28, 51-64; Sumner & Ferretti (1989) FEBS Lett. 253, 117-120]. The preferred solution conformation of [beta-Ala8]NKA(4-10) may thus be considered as a likely bioactive conformation for NK-2 selective peptides.     

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.     

New conformationally homogeneous beta-turn antagonists of the human B2 kinin receptor.

We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa(i)-D-Tic-Oic-aa(i + 3)] which adopt a type-II' beta-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i + 1 position of the beta-turn and a proline as a bridge between the C- and N-termini sides of the turn. In positions i and i + 3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i + 3, with the doubly charged analogue 49 (pKi = 6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B2 receptor antagonists.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.     

Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by 1H nuclear magnetic resonance: correlation between activities and membrane-bound conformations

Leu-enkephalin, [D-Ala2]Leu-enkephalin, and [D-Ala2]Leu-enkephalinamide (agonists) and [L-Ala2]Leu-enkephalin (inactive analogue) bind to lipid bilayer consisting of phosphatidylcholine and phosphatidylserine. The conformations that these compounds assume, once bound to perdeuterated phospholipid bilayer, have been shown to be unique, as shown by the transferred nuclear Overhauser effect (TRNOE) of 1H NMR spectroscopy. In addition, their location in the bilayer was analyzed by TRNOE in the presence of spin-labeled phospholipids. These analyses showed a clear relationship between the activity and the peptide-membrane interaction. The three active peptides, when bound to membranes, adopt the same conformation, characterized by a type II' beta-turn around Gly3-Phe4 and a gamma-turn around Gly2 (or D-Ala2). The inactive analogue, [L-Ala2]Leu-enkephalin, displayed a completely different TRNOE pattern corresponding to a different conformation in the membrane-bound state. The tyrosine residue of the active compounds is not inserted into the interior of membrane, but it is inserted into the bilayer for the L-Ala2 analogue. According to these results, [L-Ala2]Leu-enkephalin may be explained to be inactive because the mode of binding to the membranes is different from that of active compounds.     

Characterization of beta-turn and Asx-turns mimicry in a model peptide: stabilization via C--H . . . O interaction

The chemical synthesis and single-crystal X-ray diffraction analysis of a model peptide, Boc-Thr-Thr-NH2 (1) comprised of proteinogenic residues bearing an amphiphilic Cbeta -stereogenic center, has been described. Interestingly, the analysis of its molecular structure revealed the existence of a distinct conformation that mimics a typical beta-turn and Asx-turns, i.e., the two Thr residues occupy the left- and right-corner positions. The main-chain torsion angles of the N- and C-terminal residues i.e., semiextended: phi = -68.9 degrees , psi = 128.6 degrees ; semifolded: phi = -138.1 degrees , psi = 2.5 degrees conformations, respectively, in conjunction with a gauche- disposition of the obligatory C-terminus Thr CgammaH3 group, characterize the occurrence of the newly described beta-turn- and Asx-turns-like topology. The preferred molecular structure is suggested to be stabilized by an effective nonconventional main-chain to side-chain Ci=O . . . H--Cgamma(i+2)-type intraturn hydrogen bond. Noteworthy, the observed topology of the resulting 10-membered hydrogen-bonded ring is essentially similar to the one perceived for a classical beta-turn and the Asx-turns, stabilized by a conventional intraturn hydrogen bond. Considering the signs as well as magnitudes of the backbone torsion angles and the orientation of the central peptide bond, the overall mimicked topology resembles the type II beta-turn or type II Asx-turns. An analysis of Xaa-Thr sequences in high-resolution X-ray elucidated protein structures revealed the novel topology prevalence in functional proteins (unpublished). In view of indubitable structural as well as functional importance of nonconventional interactions in bioorganic and biomacromolecules, we intend to highlight the participation of Thr CgammaH in the creation of a short-range C=O . . . H--Cgamma -type interaction in peptides and proteins.     

The asparagine-stabilized beta-turn of apamin: contribution to structural stability from dynamics simulation and amide hydrogen exchange analysis

Molecular dynamics simulations of bee venom apamin, and an analogue having an Asn to Ala substitution at residue 2 (apamin-N2A), were analyzed to explore the contribution of hydrogen bonds involving Asn2 to local (beta-turn residues N2, C3, K4, A5) and global stability. The wild-type peptide retained a stable conformation during 2.4 ns of simulation at 67 degrees C, with high beta-turn stability characterized by backbone-side chain hydrogen bonds involving beta-turn residues K4 and A5, with the N2 side chain amide carbonyl. The loss of stabilizing interactions involving the N2 side chain resulted in the loss of the beta-turn conformation in the apamin N2A simulations (27 or 67 degrees C). This loss of beta-turn stability propagates throughout the peptide structure, with destabilization of the C-terminal helix connected to the N-terminal region by two disulfide bonds. Backbone stability in a synthetic peptide analogue (apamin-N2A) was characterized by NMR and amide hydrogen exchange measurements. Consistent with the simulations, loss of hydrogen bonds involving the N2 side chain resulted in destabilization of both the N-terminal beta-turn and the C-terminal helix. Amide exchange protection factors in the C-terminal helix were reduced by 9-11-fold in apamin N2A as compared with apamin, corresponding to free energy (deltaDeltaG(uf)) of around 1.5 kcal M(-1) at 20 degrees C. This is equivalent to the contribution of hydrogen bond interactions involving the N2 side chain to the stability of the beta-turn. Together with additional measures of exchange protection factors, the three main contributions to backbone stability in apamin that account for virtually the full thermodynamic stability of the peptide have been quantitated.     

Design of temperature-sensitive penicillinase repressors by replacement of Pro in predicted beta-turn structures.

Pro residues in predicted beta-turn structures were substituted with other amino acids to obtain temperature-sensitive penicillinase repressors (PenI). A mutant repressor (P70L; Pro-70 is substituted with Leu) was inactive at 48 degrees C and penP gene expression was derepressed (1,200 U/OD660 [optical density at 660 nm] ), although the mutant was still active at 30 degrees C (27 U). The heat induction ratio (penicillinase activity at 48 degrees C compared with that at 30 degrees C) of the mutant was 98 times higher than that of the wild type (i.e., 44 versus 0.45). This result indicated that the side chain of the Leu residue in P70L destroyed the proper folding of the repressor protein at the elevated temperature, whereas the Pro residue of the wild-type repressor stabilized this predicted beta-turn structure even at 48 degrees C. When the Pro residue was replaced by amino acid residues with smaller side chains (i.e., Gly and Ala), these mutant repressors were less temperature sensitive than P70L. These data suggest that the presence of the Pro residue in the beta-turn structure could be one of the key factors in stabilizing protein structure at elevated temperatures.     

A beta-turn is present in the 392-411 segment of the human fibrinogen gamma-chain. Effects of structural changes in this segment on affinity to antibody 4A5.

The interaction between fibrinogen gamma-peptide 392-411, LTIGEGQQHHLGGAKQAGDV, and monoclonal antibody 4A5, an antibody with a high affinity for both for the peptide and native fibrinogen, is being studied as a model for peptide-antibody interaction. Two-dimensional NMR studies of the free peptide at pH 5.2 indicated the presence of a significant population, about 60%, of type II beta-turn, spanning residues Gln407-Asp410. At pH 2.7, little, if any, turn structure is present. The D-Ala409 analog, which, for steric reasons, would be expected to preserve the beta-turn, and the L-Ala409 analog, which would not be expected to have this conformational feature, were synthesized, and NMR studies confirmed the respective structural predictions. The affinity of the D-Ala analog for antibody 4A5 is even greater than that displayed by native gamma 392-411, while the affinity of the L-Ala analog is less than one-tenth that of the native peptide. Both conformational and steric effects involving residues 407-410 may be important in recognition by antibody 4A5. Since gamma 392-411 includes a platelet receptor binding locus of fibrinogen, and this and related peptides are inhibitors of platelet aggregation, the D-Ala409 and L-Ala409 analogs were tested for platelet binding. Neither of the analogs displays any measurable platelet binding, indicating that the recognition requirements for the platelet receptor differ considerably from those for antibody 4A5.     

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.     

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).     

The beta-turn scaffold of tripeptide containing an azaphenylalanine residue

The conformational preferences of azaphenylalanine-containing peptide were investigated using a model compound, Ac-azaPhe-NHMe with ab initio method at the HF/3-21G and HF/6-31G(*) levels, and the seven minimum energy conformations with trans orientation of acetyl group and the 4 minimum energy conformations with cis orientation of acetyl group were found at the HF/6-31G(*) level if their mirror images were not considered. An average backbone dihedral angle of the 11 minimum energy conformations is phi=+/-91 degrees +/-24 degrees , psi =+/-18 degrees +/-10 degrees (or +/-169 degrees +/-8 degrees ), corresponding to the i+2 position of beta-turn (delta(R)) or polyproline II (beta(P)) structure, respectively. The chi(1) angle in the aromatic side chain of azaPhe residue adopts preferentially between +/-60 degrees and +/-130 degrees, which reflect a steric hindrance between the N-terminal carbonyl group or the C-terminal amide group and the aromatic side chain with respect to the configuration of the acetyl group. These conformational preferences of Ac-azaPhe-NHMe predicted theoretically were compared with those of For-Phe-NHMe to characterize the structural role of azaPhe residue. Four tripeptides containing azaPhe residue, Boc-Xaa-azaPhe-Ala-OMe [Xaa=Gly(1), Ala(2), Phe(3), Asn(4)] were designed and synthesized to verify whether the backbone torsion angles of azaPhe reside are still the same as compared with theoretical conformations and how the preceding amino acids of azaPhe residue perturb the beta-turn skeleton in solution. The solution conformations of these tripeptide models containing azaPhe residue were determined in CDCl(3) and DMSO solvents using NMR and molecular modeling techniques. The characteristic NOE patterns, the temperature coefficients of amide protons and small solvent accessibility for the azapeptides 1-4 reveal to adopt the beta-turn structure. The structures of azapeptides containing azaPhe residue from a restrained molecular dynamics simulation indicated that average dihedral angles [(phi(1), psi(1)), (phi(2), psi(2))] of Xaa-azaPhe fragment in azapeptide, Boc-Xaa-azaPhe-Ala-OMe were [(-68 degrees, 135 degrees ), (116 degrees, -1 degrees )], and this implies that the intercalation of an azaPhe residue in tripeptide induces the betaII-turn conformation, and the volume change of a preceding amino acid of azaPhe residue in tripeptides would not perturb seriously the backbone dihedral angle of beta-turn conformation. We believe such information could be critical in designing useful molecules containing azaPhe residue for drug discovery and peptide engineering.