Treatment of severe peri-implant bone loss using autogenous bone and a resorbable membrane. Case report and literature review

The objective of this study was to elucidate the solution conformation of cyclo-(1,12) Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (1) derived from the intercellular adhesion molecule-1 (ICAM-1). Cyclic peptide 1 inhibits homotypic adhesion of T-cells (Molt-3) mediated by ICAM-1 and the leukocyte function-associated antigen-1 (LFA-1) on the surface of T-cells. Cyclic peptide 1 is more potent than is the linear peptide Pen1-Pro2-Ser3-Lys4-Val5-Ile6-Leu7-Pro8-Ar g9-Gly10-Gly11-Cys12 (2) in inhibiting homotypic adhesion. The difference in biological activity of peptides 1 and 2 may be due to the more stable conformation of cyclic peptide 1 compared to linear peptide 2 or because cyclization prevents the peptide from adopting non-productive conformation. Therefore, conformational studies of cyclic peptide 1 will give a better understanding of its biological active conformation. The conformational studies of cyclic peptide 1 were done by NMR, CD and molecular dynamics simulations. NMR studies indicated that the major conformation of cyclic peptide 1 contained trans-configuration at both X-Pro peptide bonds. Type I beta-turns at Lys4-Val5-Ile6-Leu7 and Leu7-Pro8-Arg9-Gly10 were found in cyclic peptide 1. The C- and N-terminal regions of this peptide were stabilized by antiparallel beta-sheet-like structure with the presence of intramolecular hydrogen bonds. The overall structure of this peptide exposed the hydrophobic side chains on one face of the molecule and the hydrophilic side chains on the other.     

Structure-function analysis of a series of glucagon-like peptide-1 analogs

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1.) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely, from an examination of the amino acid sequence and from previously published observations, to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a predetermined 3D structure. Constraint was achieved by the introduction of disulfide bonds and specific side chain-side chain cross-links. The biological relevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F cells and to elicit a functional response, cyclic AMP production. NMR solution structures were obtained for the most biologically relevant of these analogs. The results of this study indicated that the residues necessary for the biological activity of GLP-1 occupy approximately three equally-spaced regions of the peptide 3D structure, at the corners of an equilateral triangle whose sides are, at a minimum estimate, 12-15A.     

The conformation of peptide thymosin alpha 1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin alpha1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin alpha1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin alpha1 has a structure characterized by two regions. A beta-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an alpha helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin alpha1.     

Circadian rhythm of intraocular pressure in the rat

In order to investigate the relationship between the bioactive conformation of a peptide and its set of thermodynamically accessible structures in solution, the conformational profile of the tetrapeptide Ac-Pro-Ala-Pro-Tyr-OH was characterized by computational methods. Search of the conformational space was performed within the molecular mechanics frame-work using the AMBER4.0 force field with an effective dielectric constant of 80. Unique structures of the peptide were compared with its bioactive conformation for the protein Streptomyces griseus Protease A, as taken from the crystal structure of the enzyme-peptide complex. The results show that the bound conformation is close to one of the unique conformations characterized in the conformational search of the isolated peptide. Moreover, the lowest energy minimum characterized in the conformational search exhibits large deviations when compared to the bound conformation of the crystal structure.     

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.     

Conformational changes in a mammalian voltage-dependent potassium channel inactivation peptide

Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (Kv) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic Kv3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no alpha-helix or beta-sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial beta-sheet structure. Oxidation of the Kv3.4 ball peptide facilitates formation of a disulfide bond between Cys6 and Cys24 and adoption of a partial beta-sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt beta-sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the Kv3.4 ball peptide structure predicts a beta-hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant Kv3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt beta-structure whatever the environment, and regardless of oxidation state. The results suggest that the Kv3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the Kv3.4 ball domain in vivo.     

Conformational preferences of a peptide corresponding to the major antigenic determinant of foot-and-mouth disease virus: implications for peptide-vaccine approaches

The conformational preferences in solution of a peptide corresponding to the GH loop of the VP1 capsid protein from the foot-and-mouth disease virus were examined by proton nuclear magnetic resonance and circular dichroism. The GH loop is the major antigenic determinant of the virus and participates in cell attachment through an integrin-like Arg-Gly-Asp sequence. The synthetic peptide, corresponding to residues Gly132 to Ser162 of the VP1 capsid protein of the serotype O, is largely disordered in aqueous solution as shown by the absence of long- and medium-range NOE contacts and by random-like chemical shifts values. Helical contents in aqueous solution were estimated to be less than 10%, as determined by extrapolation of trifluoroethanol titration from CD measurements, in good agreement with estimations from NMR experiments. In the presence of 40% trifluoroethanol an alpha-helix, flanked by two proline residues between Asn12 (Asn143 in the intact protein) and Leu28 (159), is induced. This contrasts with the 3(10) helix observed between residues Leu148 and Val155 in the crystal structure of the dithiothreitol-reduced virus, indicating that the cosolvent does not stabilize a residual, low-populated structure, similar to that in the intact virus. Several algorithms also fail to predict the structure found in the intact virus because these are based mainly on local sequence information. The lack of structure of the peptide in aqueous solution strongly suggests that the conformational determinants sufficient for the structure stabilization of this highly immunogenic antigen are mostly dictated by interactions of the loop with other regions of the virus structure, and do not arise from local amino acid sequence information. The ability of designed GH-VP1 peptides to neutralize anti-virus antibodies is likely to arise from antibody-induced conformation of the peptide and its application as peptide vaccines is not straightforward. Similarly, insertion of these peptides in carriers or macromolecular assemblies as vaccine vectors would depend on the conformation adopted at the insertion site and its success cannot be predicted.     

Chronic neurologic effects of pesticide overexposure

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of a peptide with a 20-amino-acid sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. A peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) was found to inhibit syncytia formation after virus infection and after transfection of Cos cells with the HN (hemagglutinin-neuraminidase) and F protein cDNAs. Using an F protein gene that requires addition of exogenous trypsin for cleavage, it was shown that the peptide exerted its inhibitory effect prior to cleavage. The three-dimensional conformation of the peptide in aqueous solution was determined through the use of NMR and molecular modeling. Results showed that the peptide formed a helix with properties between an alpha-helix and a 3(10)-helix and that leucine residues aligned along one face of the helix. Side chain salt bridges and hydrogen bonds likely contributed to the stability of the peptide secondary structure. Analysis of the aqueous solution conformation of the peptide suggested mechanisms for specificity of interaction with the intact F protein.     

Structure/antigenicity relationship of cyclic and linear peptides mimicking the V3 loop of HIV2 envelope glycoprotein

We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K0.5). The K0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.     

Primary active transport of peptidic endothelin antagonists by rat hepatic canalicular membrane

The biliary excretion mechanism of three derivatives of BQ-123, an anionic cyclopentapeptide, was examined using isolated canalicular membrane vesicles (CMVs) from Sprague-Dawley rats. The uptake by CMV of BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, was stimulated by ATP. An "overshoot" phenomenon and saturation were observed for the ATP-dependent uptake of these three peptides. The Michaelis-Menten constants (Km) for the uptake of BQ-485 and BQ-518 were comparable to the inhibition constants (Ki) for their inhibitory effects on ATP-dependent [3H]BQ-123 uptake. The uptake of BQ-485 showed the highest value and was inhibited by BQ-123 with a Ki that was comparable to the Km for BQ-123 uptake. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 was much lower in CMVs from Eisai hyperbilirubinemic rats, a strain having a hereditary defect of the canalicular multispecific organic anion transporter (cMOAT). These results suggest that both BQ-485 and BQ-518 principally share the cMOAT transporter with BQ-123. Compound A almost completely inhibited BQ-123 uptake, although its ATP-dependent uptake was much lower than that of the other three peptides. The ATP-dependent uptake of compound A was not very different in Sprague-Dawley rats and Eisai hyperbilirubinemic rats and was not inhibited by S-(2, 4-dinitrophenyl)-glutathione, a typical substrate for cMOAT. Thus, although compound A inhibits cMOAT-mediated transport, its own transport by cMOAT is minimal and mediated by another transporter. This low degree of primary active transport by cMOAT may be the principal reason for its relatively longer residence in the circulation.     

Conformational studies by NMR of the antimicrobial peptide, drosocin, and its non-glycosylated derivative: effects of glycosylation on solution conformation

Drosocin is a cationic 19 amino acid peptide secreted by Drosophila in response to septic injury. The sequence (GKPRPYSPRPTSHPRPIRV) contains six Pro and four Arg residues which are incorporated into three repeated triplet sequences Pro-Arg-Pro. The peptide is glycosylated at Thr11 and has potent antimicrobial activity. This activity is markedly reduced on deglycosylation, but a structural basis for this has not been previously established. In the current study, the solution conformations of drosocin and its non-glycosylated derivative were determined by NMR spectroscopy and structure calculations. The NMR and structure studies showed that the peptides have significant populations of essentially random coil conformations in aqueous solution. Addition of 50% trifluoroethanol causes the development of small populations of folded conformations, mainly in the form of turns. In particular, turn elements occur near residues 4-7, 10-13, 17, and 18. No substantial difference was detected in the predominantly random coil conformation of the glycosylated and non-glycosylated forms, but there are subtle differences in the small populations of folded conformers. In particular, the turn at residues 10-13 tends toward a more extended structure on glycosylation, while there is some tightening of the downstream turn at residues 17 and 18. There are a significant number of nuclear Overhauser enhancement contacts between the sugar moiety and the peptide near the glycosylation site, consistent with a close association between them. Despite this close association, the pKa of H13, which is proximate to the glycosylation site, was found to be unaffected by glycosylation.     

Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product beta-cyclodextrin

Crystals of the inactive mutant Glu257-->Ala of cyclodextrin glycosyltransferase were soaked with the cyclodextrin (CD) derivative S-(alpha-D-glucopyranosyl)-6-thio-beta-CD. The structural analysis showed its beta-CD moiety with no density indication for the exocyclic glucosyl unit. For steric reasons, however, the position of this unit is restricted to be at only two of the seven glucosyl groups of beta-CD. The analysis indicated that the enzyme can cyclize branched alpha-glucans. The ligated beta-CD moiety revealed how the enzyme binds its predominant cyclic product. The conformation of the ligated beta-CD was intermediate between the more symmetrical conformation in beta-CD dodecahydrate crystals and the conformation of a bound linear alpha-glucan chain. Its scissile bond was displaced by 2.8 A from the position in linear alpha-glucans. Accordingly, the complex represents the situation after the cyclization reaction but before diffusion into the solvent, where a more symmetrical conformation is assumed, or the equivalent state in the reverse reaction. Furthermore, a unifying nomenclature for oligosaccharide-binding subsites in proteins is proposed.     

Cyclopropane-derived peptidomimetics. Design, synthesis, evaluation, and structure of novel HIV-1 protease inhibitors

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.     

A new approach to secondary structure evaluation: secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover alpha-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing alpha-helical structures. For segments with dominating beta-sheet conformation, however, the application of this method is limited due to the stability and clustering of beta-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed beta-sheet CD signals. Nevertheless, the results of this method especially for alpha-helical segments are very impressive. All alpha-helical and 71% of the beta-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of alpha-helical secondary structure elements and help localizing them on the sequence string.     

The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites

The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution. The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII). A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII. These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity. The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site.     

Serum sensitivity and cell surface hydrophobicity of Klebsiella pneumoniae treated with gentamicin, tobramycin and amikacin

The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 degrees C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.     

Structural model of a cyclic dynorphin A analog bound to dodecylphosphocholine micelles by NMR and restrained molecular dynamics

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.     

Role of Epstein-Barr virus in fine-needle aspirates of metastatic neck nodes in the diagnosis of nasopharyngeal carcinoma

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1),a = 11.843(2), b = 21.493(4), c = 26.676(4) A3 and V = 6790 A3. Data were collected on a CAD4 diffractometer using MoK alpha radiation (lambda = 0.7107 A) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystal-lographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val2 residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state.     

Conformational studies of a synthetic peptide from the putative lipid-binding domain of bovine milk component PP3

In bovine milk, a glycosylated phosphoprotein, component PP3, is known for its remarkable emulsifying properties and its capability to inhibit lipolytic activities. The determination of its primary structure is not sufficient to explain these properties. Secondary structure predictions of component PP3 and of its homologous proteins were achieved using a combination of multiple predictive methods. Based on this study, the f 119-135 region of component PP3 was proposed to be likely to adopt an amphipathic helical conformation, which is a lipid-binding motif. The conformation of the synthetic peptide corresponding to the C-terminal f 119-135 part of bovine component PP3 was analyzed by circular dichroism experiments using various media. The circular dichroism data indicated that the peptide was able to form an amphipathic alpha-helix structure in trifluoroethanol as well as in the presence of sodium dodecyl sulfate or acidic and neutral lipids, but not in water. Moreover, the conformation of this peptide is solvent dependent because it was found to adopt a beta-sheet structure for low concentrations of sodium dodecyl sulfate or a low molar ratio of acidic lipid to peptide. Tensiometric measurements showed that the amphipathic C-terminal region of component PP3 is highly tensioactive and, thus, must be responsible for the particular behavior of the protein in emulsions.     

Structure-activity relationships and physico-chemical properties of synthetic lipopeptide inhibitors of PKC

Four synthetic lipopeptides, (K-pm 19,31), (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) with the lysine-palmitoyl (K-pm) residue as a lipophilic moiety, based on the pseudosubstrate sequence 19RFARKGALRQKNV31 (R19-V31), were found to be potent protein kinase C (PKC) inhibitors. However, the lipopeptides (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) were also found to act as protein kinase cAMP-dependent (PKA) inhibitors. Peptide (K-pm 19,31), the least water soluble, is marginally selective towards PKC, unlike the other palmitoyl derivatives studied here. Since the non-palmitoylated analogues (K 19,31), (K-ac 19,31), (K 19,21,31) and (K-ac 19,21,31) were inhibitors of PKC but not of PKA, the palmitoyl moiety must play a role in the specificity of protein kinase inhibition. In vitro, the lipophilic peptides showed greater stability to protease-mediated hydrolysis than the pseudosubstrate peptide depending upon the number of lipophilic (K-pm) residues. CD studies showed that in comparison with the peptide analogues, the remarkable resistance of the pseudosubstrate (R19-V31) to adopt an alpha-helix conformation in TFE, known to be strongly alpha-helix inducing, rules out this structure as the peptide binding conformation to PKC. By contrast, in aqueous media all the peptides show an extended conformation that correlates well with their inhibitory activity. This is in compliance with the crystallographic observation that an extended structure has been observed for the (5-24) PKI peptide inhibitor bound to PKA.