Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part II: Studies in the SDS micelle.

There is much evidence to support the hypothesis that lipids play a role in the interaction of peptide hormones with their membrane receptors. This interaction through change of peptide conformation can facilitate the entry of the hormone into the microenvironment of the receptor. In the present study we have examined the interaction of vasopressin and mesotocin with a lipid-sodium dodecylsulfate (SDS) micelle-using 2D nuclear magnetic resonance (NMR) and theoretical methods. Solution structures of two hormones in solution with SDS were established using the nuclear Overhauser effect (NOE) and the (3)J(NHHalpha) couplings. The amino acid sequences of these peptides are: c[C(1)-Y(2)-F(3)-Q(4)-N(5)-C(6)]-P(7)-R(8)-G(9)-NH(2) ([Arg(8)]vasopressin, AVP) and c[C(1)-Y(2)-I(3)-Q(4)-N(5)-C(6)]-P(7)-I(8)-G(9)-NH(2) (MT). Each of the peptides was found to occur as one stable conformation. AVP adopts the cis configuration on the Cys(1)-Tyr(2) peptide bond, a finding not reported so far. The three-dimensional structures of the two peptides studied were determined by a method that consisted of time-averaged molecular dynamics in an explicit SDS micelle with the parm99 force field in AMBER8.0 package. All calculated structures of the studied peptides form beta-turns in their cyclic parts. The C-terminal fragment of MT is bent, whereas that of AVP is extended.     

Oxytocin antagonist blocks the vasodepressor but not the vasopressor effect of neurohypophysial peptides in chickens

Cockerels with permanent cannulas in the brachial artery and vein were put into isolated slings. Arterial pressure and heart rate were continuously recorded. Following habituation, tests were initiated. In each cockerel 2 nmol/kg of the tested neurohypophysial peptide (NPs) or analogue was IV injected six times at 6-min intervals. Arginine vasotocin (AVT) caused an immediate vasodepressor (VDP) effect and tachycardia. These subsided within 20–30 s and were followed by a vasopressor (VP) response and bradycardia. On repeated injections of AVT, the VDP response declined and bradycardia intensified. Arginine vasopressin (AVP), oxytocin (OT), and mesotocin (MT) had short-lasting VDP effect in the following order of potency: OT = MT > AVT > AVP. Only AVT and, more effectively, AVP, caused a VP response. The VDP effect of MT and OT declined on repeated injections. When AVT was injected after three injections of MT, it had mostly an immediate VP effect. Although the V₁ agonist is VP in chickens, at the dose used the V₁ antagonist, [d(CH₂)₅,O-Me-Tyr²]AVP, had no effect on cardiovascular responses to AVT. Pretreatment with OT antagonist, [d(CH₂)₅-O-Me-Tyr^2,Thr^4,Tyr^9,Orn⁸]VT, abolished the VDP effect of all NPs. Thus, MT had no effect on blood pressure, whereas AVP and, more effectively, AVT, had a marked immediate VP action. In chickens the VDP effect of NPs is probably mediated by an OT/MT-like receptor, wherein the peptide's ring structure, shared by AVT, OT, and MT, is important. The VP effect is mediated by a receptor only partially similar to the mammalian V₁ receptor, where arginine in position 8, shared only by AVT and AVP, is necessary for action, and the native AVT is more effective than the mammalian AVP. This receptor reacts to the V₁ agonist but probably not to the V₁ antagonist.     

Solution and solid state conformational preferences of a family of cyclic disulphide bridged tetrapeptides

A set of cyclic tetrapeptides of the general form cyclo (Boc‐Cys‐Pro‐ X ‐Cys‐OMe) with X being L‐ / D‐Ala , L‐ / D‐Val , and L‐ / D‐Trp was synthesized. These peptides serve as model systems for structure elucidation in solution and feature a variety of structural motifs — namely a β‐turn with intramolecular hydrogen bonding interactions, cis/trans isomerism, and a disulphide bond. In this work, we performed a comprehensive structural analysis focussing on their β‐turn conformational preferences using NMR, VCD, and Raman spectroscopy. Our results provide evidence for a strong influence of a single stereocenter on the structures of the peptides whereas solvent polarity does not significantly affect them. Additionally, the solid state conformational preferences were studied by crystal structure analysis. Overall, a general trend for the conformational preferences of this set of peptides can be concluded from the results of the complementary investigations.     

Analogs of arginine vasopressin modified in the N-terminal part of the molecule with a conformationally constrained cis-peptide bond motif.

The present work is part of our studies aimed at clarifying the influence of steric constraints in the N-terminal part of arginine vasopressin (AVP) and its analogs on the pharmacological activity of the resulting peptides. We describe the synthesis of eight new analogs of AVP or [3-mercaptopropionic acid (Mpa)]AVP (dAVP) substituted at positions 2 and 3 or 3 and 4 with two diastereomers of 4-aminopyroglutamic acid. The steric constraints provided by this modification turned out, however, so strong that all the peptides were inactive in all of the bioassays (pressor, antidiuretic and uterotonic tests).     

The regulation of the corticomelanotropic cell activity in aves. III--Effect of various peptides on the release of MSH from dispersed, perfused duck pituitary cells. Cosecretion of ACTH with MSH

1. The melanotropin-releasing activity of arginine-vasopressin (AVP), arginine-vasotocin (AVT), oxitocin (OT), mesotocin (MT) and corticotropin-releasing factor (CRF) was studied in the duck using dispersed, perfused pituitary cells and a specific alpha-MSH RIA. 2. Log dose-response curves were obtained for all the peptides ranging from 5 to 100 ng/ml. All peptides behaved as partial agonists compared to duck median eminence extracts (DME). 3. AVT and MT displayed an alpha-MSH releasing capacity of 60% relative to DME whereas all other peptides behaved as weak agonists with less than 15% capacity relative to DME. 4. AVT and CRF when perfused together acted synergistically on alpha-MSH release yielding a dose response line whose slope approximated that of DME. 5. ACTH was cosecreted together with alpha-MSH in all situations studied with an ACTH to alpha-MSH molar ratio of about 10. 6. It is concluded that CRF and neurohypophyseal peptides may be physiological stimulators of both alpha-MSH and ACTH release in aves.     

Theoretical conformational analysis of six arginine vasopressin analogs with the L-naphthylalanine in position 3.

Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with L-2-Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (L-1-Nal)3, (D-Arg)8] VP and [Mpa1, (L-2-Nal)3, (D-Arg)8] VP are potent and selective V2 agonists. Moreover [(L-2-Nal)3, (D-Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(L-1-Nal)3] AVP, [(L-2-Nal)3] AVP, [(L-1-Nal)3, (D-Arg)8] VP, [(L-2-Nal)3, (D-Arg)8] VP, [Mpa1, (L-1-Nal)3, (D-Arg)8] VP, [Mpa1, (L-2-Nal)3, (D-Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low-energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5 and Gln4-Asn5, respectively. The conformation at the Gln4-Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

Analogues of arginine vasopressin modified in position 2 and 3 with conformationally constrained dipeptide fragments.

This study describes the synthesis and some pharmacological properties of ten new analogues of arginine vasopressin (AVP) containing a conformationally constrained dipeptide fragment in the N-terminal part of their molecules. Amino acid residues in positions 2 and 3 of AVP and some of its agonistic analogues were replaced with -Phe-Phe and D-Phe-D-Phe, dipeptides having a -CH2-CH2- link bridging two nitrogens. All the new peptides were tested for vasopressor and antidiuretic activities. Four peptides with pA2 values ranging from 5.96 to 7.21 turned out to be weak or moderately potent V1a antagonists. The results supplied new information about the structure-activity relationship of AVP analogues. As some of these were unexpected, they point to the need for caution when extrapolating previously known effects of modifications to analogues having conformationally constrained fragments in their molecules.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Diabetes Increases the Expression of Hypothalamic Neuropeptides in a Spontaneous Model of Type I Diabetes, the Nonobese Diabetic (NOD) Mouse

1. Synthesis of oxytocin (OT) and arginine-vasopressin(AVP) is increased in induced models of Type I diabetes, such as thestreptozotocin model. However, these parameters have not yet been evaluated inspontaneous models, such as the nonobese diabetic mouse (NOD). Therefore, we studied in the magnocellular cells of the paraventricular nucleus (PVN)of nondiabetic and diabetic 16-week-old female NOD mice and control C57Bl/6mice, the immunocytochemistry of OT and AVP peptides and their mRNA expression, using nonisotopic in situ hybridization (ISH).2. In nondiabetic and diabetic NOD female mice, the number of OT- and AVP-immunoreactive cells were similar to those of the controls, whereas immunoreaction intensity was significantly higher for both peptides in diabetic NOD as compared with nondiabetic NOD and control C57Bl/6 mice.3. ISH analysis showed that the number of OT mRNA-containing cells was in the same range in the three groups, whereas higher number of AVP mRNA expressing cells was found in diabetic NOD mice. However, the intensity of hybridization signal was also higher for both OT and AVP mRNA in the diabetic group as compared with nondiabetic NOD and control mice.4. Blood chemistry demonstrated that haematrocrit, total plasma proteins, urea, sodium, and potassium were within normal limits in diabetic mice. Thus, NODmice were neither hypernatremic nor dehydrated.5. We suggest that upregulation of OT and AVP reflects a high-stress condition in the NOD mice. Diabetes may affect neuropeptide-producing cells of the PVN, with the increased AVP and OT playing a deleterious role on the outcome of the disease.     

Prediction of antiviral peptides derived from viral fusion proteins potentially active against herpes simplex and influenza A viruses

There are very few antiviral drugs available to fight viral infections and the appearance of viral strains resistant to these antivirals is not a rare event. Hence, the design of new antiviral drugs is important. We describe the prediction of peptides with antiviral activity (AVP) derived from the viral glycoproteins involved in the entrance of herpes simplex (HSV) and influenza A viruses into their host cells. It is known, that during this event viral glycoproteins suffer several conformational changes due to protein-protein interactions, which lead to membrane fusion between the viral envelope and the cellular membrane. Our hypothesis is that AVPs can be derived from these viral glycoproteins, specifically from regions highly conserved in amino acid sequences, which at the same time have the physicochemical properties of being highly exposed (antigenic), hydrophilic, flexible, and charged, since these properties are important for protein-protein interactions. For that, we separately analyzed the HSV glycoprotein H and B, and influenza A viruses hemagglutinin (HA), using several bioinformatics tools. A set of multiple alignments was carried out, to find the most conserved regions in the amino acid sequences. Then, the physicochemical properties indicated above were analyzed. We predicted several peptides 12-20 amino acid length which by docking analysis were able to interact with the fusion viral glycoproteins and thus may prevent conformational changes in them, blocking the viral infection. Our strategy to design AVPs seems to be very promising since the peptides were synthetized and their antiviral activities have produced very encouraging results.     

Hallmarks of molecular action of microtubule stabilizing agents: effects of epothilone B, ixabepilone, peloruside A, and laulimalide on microtubule conformation

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αβ-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.     

靶向膜型1基质金属蛋白酶反义肽的虚拟筛选与分子模拟

膜型1基质金属蛋白酶(Membrane type-1 matrix metalloproteinase,MT1-MMP,MMP14)在肿瘤的发生发展及转移中起着重要作用,是肿瘤潜在理想的药物靶标。为了获得MT1-MMP结合肽,我们首先采用生物信息学方法分析MMPs序列,获得MT1-MMP差异大且特异的序列。以此为正义肽靶标,设计反义肽库,然后通过分子对接、分子动力学模拟以及体外细胞实验等多种方法,进行靶向MT1-MMP反义肽的筛选与活性研究。多序列比对确定了位于MT1-MMP环区的特异序列AYIREGHE(简称MT1-loop),并构建1 536条反义肽。经两轮虚拟筛选,选取打分位于前五的反义肽用于后续研究。该五条反义肽与MT1-MMP存在较强的相互作用且能很好地对接于正义肽区域。进一步分析其与MMPs其他家族成员(MMP1-3,MMP7-13,MMP14HPX,MMP16)的亲和力,发现反义肽FVTFPYIR对MT1-MMP具有更强的特异性。分子动力学模拟表明,反义肽FVTFPYIR可能是通过影响受体MT1-MMP的构象稳定性,进而影响其功能活性。体外细胞实验初步确定反义肽FVTFPYIR可选择性地抑制表达MT1-MMP的人成骨肉瘤细胞MG63和乳腺癌MDA-MB-231细胞的增殖。本研究为抗肿瘤反义肽先导药物的研发提供了一种新的思路与途径。     

Conformations of bombolitins I and III in aqueous solutions: circular dichroism, 1H NMR, and computer simulation studies

The heptadecapeptides bombolitin I and bombolitin III are two of a series of peptides postulated to be biologically active within a membrane environment. In the preceding paper [Bairaktari, E., Mierke, D.F., Mammi, S., & Peggion, E. (1990) Biochemistry (preceding paper in this issue)] the conformational preferences of these peptides in the presence of SDS surfactant micelles, a mimetic for biological membranes, were examined. During these studies the conformations of these peptides were investigated in aqueous solutions by circular dichroism and nuclear magnetic resonance. A large difference was observed for the two peptides. Bombolitin I lacks any observable secondary structure in aqueous solution, independent of temperature, pH, and concentration. In striking contrast, bombolitin III adopts a well-defined alpha-helix at concentrations greater than 1.3 mM. This is indeed surprising given the great similarity of the two peptides. The alpha-helix of bombolitin III is pH dependent, with a great decrease in the observed secondary structure at pH values below 3.5. This observation could only be due to the protonation of the Asp residue at the fifth position. These findings suggest that the secondary structure arises from molecular aggregation of bombolitin III through the formation of a salt bridge involving the Asp side chain. The alpha-helix observed at "high" concentration (greater than 2.5 mM) has been characterized by CD and by the NOE's measured throughout a majority of the peptide. The experimentally determined structure has been energy refined with restrained molecular dynamics. The conformational results from this study are then compared with the conformations found in the presence of surfactant micelles.     

The regulation of the corticomelanotropic cell activity in Aves--II. Effect of various peptides on the release of ACTH from dispersed, perfused duck pituitary cells

We have estimated the corticotropin-releasing activity (CRA) of different neurohypophyseal peptides and synthetic corticotropin-releasing factor (CRF) in the duck, using perfused dispersed pituitary cells and an ACTH radioimmunoassay adapted to duck material. Log dose-response curves were obtained for different doses of arginine-vasopressin (AVP), arginine-vasotocin (AVT), mesotocin (MT), oxitocin (OT) and ovine CRF (oCRF) and compared to the response obtained with dilutions of duck median eminence extracts (DME). All peptides tested behaved as partial agonists compared to DME. AVT and MT were the most potent of all peptides tested, with a capacity of 60% relative to DME. CRF was a weak agonist together with AVP and OT. AVT and CRF perfused together at equal doses significantly potentiated the effect of each other, yielding a dose-response line whose slope approximated that of DME. A similar design was used to test the CRA of the same substances in the rat. The main difference in the pattern of response between the two species was the low potency displayed by all the neurohypophyseal peptides in the rat, compared with CRF which, in contrast with what occurred with the duck system, was the most potent secretagogue of all peptides tested. It is concluded that in birds, as in mammals, the control of ACTH secretion may be exerted by neurohypophyseal peptides and a CRF-like peptide acting synergistically upon the corticomelanotropic cell.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

Induced conformational states of amphipathic peptides in aqueous/lipid environments.

Specific conformational effects have been reported for amphipathic model peptides upon binding of defined hydrophobic domains to nonpolar stationary phases during reversed-phase high performance liquid chromatography (RP-HPLC). Such induced conformations are found to be especially pronounced for peptides that are amphipathic in an alpha-helical conformation. Such induced amphipathic conformations resulted in substantially later elution than predicted using amino acid-based retention coefficients. In the present study, the induced conformational behavior of model peptides observed during RP-HPLC was correlated with their secondary structure as determined by circular dichroism (CD) spectroscopy in both aqueous solution and C18-mimetic environments. The experimental retention times of the peptides studied were found to correlate with their CD spectra in the presence of lipids, whereas a poor correlation was observed with their CD spectra in the presence of trifluoroethanol. A new approach was developed to evaluate the induction of secondary structure in peptides due to interactions at aqueous/lipid interfaces, which involves the measurement of the CD ellipticities of peptides bound to a set of C18-coated quartz plates. An excellent correlation was found in this environment between the RP-HPLC retention times and CD ellipticities of the bound peptides.     

Pituitary and adrenal responses to ovine corticotropin releasing factor and vasopressin injected into young and adult guinea-pigs

Comparison of effects of synthetic ovine corticotropin releasing factor (oCRF), Arginine-Vasopressin (AVP) and the combination of both peptides have been tried in adult and 7-days-old guinea-pigs. On plasmas collected 15 min after interscapulary injection, cortisol, aldosterone and ACTH were measured. The different circulating forms of ACTH were isolated by Sephadex G50 column chromatography, with 1% formic acid and measured by radioimmunoassay. Thus, in the guinea-pig plasma, we detected three immunoreactive forms of ACTH: a "big" molecular form (Mr greater than 20000), an "intermediate" (Mr = 9500) and a "little" ACTH form (Mr = 4500) which was eluted in the same fractions as human 1-39 ACTH. In adult guinea-pigs, CRF increased total ACTH and the "intermediate" form and also plasma cortisol concentrations whereas AVP remained without significant effect excepted a rise in cortisol levels. Injected together, CRF and AVP enhanced plasma concentrations of total ACTH, of the three circulating forms and of cortisol. In 7-days-old guinea-pigs, both CRF and AVP increased plasma concentrations of total, of "intermediate" ACTH and of cortisol and aldosterone whereas the combination of both peptides enhanced dramatically plasma concentration of total ACTH suggesting a magnifying effect of AVP on CRF activity still more efficient in young than in adult guinea-pigs.     

Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.     

A genetic algorithm with conformational memories for structure prediction of polypeptides

We have developed an iterative hybrid algorithm (HA) to predict the 3D structure of peptides starting from their amino acid sequence. The HA is made of a modified genetic algorithm (GA) coupled to a local optimizer. Each HA iteration is carried out in two phases. In the first phase several GA runs are performed upon the entire peptide conformational space. In the second phase we used the manifestation of what we have called conformational memories, that arises at the end of the first phase, as a way of reducing the peptide conformational space in subsequent HA iterations. Use of conformational memories speeds up and refines the localization of the structure at the putative Global Energy Minimum (GEM) since conformational barriers are avoided. The algorithm has been used to predict successfully the putative GEM for Met- and Leu-enkephalin, and to obtain useful information regarding the 3D structure for the 8mer of polyglycine and the 16 residue (AAQAA)(3)Y peptide. The number of fitness function evaluations needed to locate the putative GEMs are fewer than those reported for other heuristic methods. This study opens the possibility of using Genetic Algorithms in high level predictions of secondary structure of polypeptides.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N-terminal part of the molecule with l-beta-homophenylalanine.

In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l-beta-homophenylalanine (beta-Hph). Two of them had just this single modification, the next two peptides are analogues of the V2 agonist, namely [3-mercaptopropionic acid (Mpa)1]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP, with beta-Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa(1),beta-Hph2]AVP, [Cpa1,beta-Hph2]AVP, [Cpa1,beta-Hph3]AVP, turned out to be uterotonic antagonists with pA2 = 6.3 +/- 0.2, 6.3 +/- 0.1, 6.0 +/- 0.3 respectively. The last one exhibited antipressor properties also (pA2 = 6.4 +/- 0.1).