The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

In vivo structure-activity studies on the dark-color-inducing neurohormone of locusts.

In the 11-residue long dark-color-inducing neurohormone (DCIN = [His7]-corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by D-phenylalanine (D-Phe). The dark-color-inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN-deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by D-Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence -Ser-Xxx-Gly-Trp- (Xxx = His in theDCIN) that seems to be important for the dark-color-inducing activity, as found also in another study (Insect Biochem. Mol. Biol.32, 2002, 909). GIn4, however, is outside of this partial sequence.Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the -Ser-Xxx-Gly-Trp- partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8was substituted by D-Phe or by L-Phe. Therefore the -Ser-Xxx-Gly-Trp- partial sequence does not seem to be stabilized by a type II beta-turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

In vivo structure-activity studies on the dark-color-inducing neurohormone of locusts.

In the 11-residue long dark-color-inducing neurohormone (DCIN = [His7]-corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d-phenylalanine (d-Phe). The dark-color-inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN-deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d-Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence -Ser-Xxx-Gly-Trp- (Xxx = His in the DCIN) that seems to be important for the dark-color-inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the -Ser-Xxx-Gly-Trp- partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d-Phe or by l-Phe. Therefore the -Ser-Xxx-Gly-Trp- partial sequence does not seem to be stabilized by a type II beta-turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures

Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.     

Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6–13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8–13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.     

Tryptic digestion of peptides corresponding to modified fragments of human growth hormone-releasing hormone

The objective of this study was to examine the degradation of short peptides corresponding to modified fragments of human growth hormone-releasing hormone by trypsin. Six analogues of pentapeptide 9-13 of human growth hormone-releasing hormone containing homoarginine, ornithine, glutamic acid, glycine, leucine or phenylalanine residue in position 11, two analogues of hexapeptide 8-13 of human growth hormone-releasing hormone and two analogues of heptapeptide 7-13 of human growth hormone-releasing hormone containing homoarginine or glycine residue in position 11 were obtained. The peptides were subjected to digestion by trypsin and the course of reaction was monitored using HPLC. It was found that the rate of hydrolysis of the Lys(12)-Val(13) peptide bond depends on the amino-acid residue preceding Lys(12). The extension of the peptide chain towards the N-terminus by introduction of consecutive amino-acid residues corresponding to the human growth hormone-releasing hormone sequence accelerates the hydrolysis process. These results may be of assistance in designing new analogues of human growth hormone-releasing hormone, more resistant to the activity of proteolytic enzymes.     

Conformational flexibility and biological activity of salmon calcitonin

We have assessed the biological activity of salmon calcitonin I (sCT) using an in vivo biological assay of hypocalcemic activity in rats. The changes in biological activity observed are explained on the basis of changes in the conformational properties of the hormone analogues. Helical content in the presence and absence of lipids and detergents was assessed by using circular dichroism, and the section of the molecule that folds into a helix was predicted on the basis of the helix-coil transition theory of Mattice and co-workers. In the amino acid sequence of sCT, residue 8 is valine and residue 16 is leucine. The synthetic calcitonin derivatives [Gly8]sCT and [Ala16]sCT have higher biological activity than the native hormone although they have a lower helical content. The increased biological activity of these derivatives is ascribed to an increase in their conformational flexibility resulting from the substitution of amino acid residues with less bulky side chains and less tendency to form helical structures. The derivative [Met8]sCT has less substitution than sCT on the beta-carbon at position 8, but it has increased helix-forming potential in the region of residues 8-12. These two factors affect conformational flexibility in opposite ways, resulting in the biological activity of [Met8]sCT being slightly higher than that of sCT. However, increased conformational flexibility does not always increase biological activity. Substitution of the L-arginine at residue 24 for a D-arginine has little effect on the conformational properties or biological activity of sCT. However, [Gly8, D-Arg24]sCT is less active than sCT, [Gly8]sCT, or [D-Arg24]sCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.     

Neuroendocrine control of thymic hormonal production. II. Stimulatory effects of endogenous opioids on thymulin production by cultured human and murine thymic epithelial cells

Data have now accumulated to strongly demonstrate that several neuropeptides, including endogenous opioids, can have immunomodulatory functions. Most of the studies have so far focused on the direct action of these substances on lymphocytes. We decided to investigate whether thymic epithelial cells (TEC) - the major component of the thymic microenvironment - could also be modulated by endogenous opioids. Primary cultures of human and murine TEC were subjected to several opioids (alpha-beta- or gamma-endorphins, as well as met- or leuenkephalins) applied in concentrations ranging from 10(-6) to 10(-9) M. On the following days we measured the levels of thymulin (a chemically-defined thymic hormone known to stimulate some steps of T-cell differentiation) in the culture supernatants, as well as the numbers of thymulin containing cells, evaluated by immunofluorescence with an anti-thymulin monoclonal antibody. After treatment of TEC cultures with beta-endorphin or leu-enkephalin a significant increase in the levels of thymulin in the culture media was observed, paralleled by a rise in the percentage of thymulin containing cells. In addition, this stimulatory effect was dose-dependent. Preincubation of the opioids with the specific antibodies abrogated the opioid-induced stimulatory effect on TEC. Moreover, naloxone, an opioid receptor antagonist, blocked the effect of beta-endorphin on thymulin production, suggesting that the effect of this neuropeptide on epithelial cells was mediated by an opioid receptor. Importantly, no effect on thymulin production was observed with the other opioids used, whatever the dose. These results suggest that, at least in vitro, beta-endorphin and leu-enkephalin stimulate the hormonal function of the thymic epithelium. These findings lead to the general concept that the modulatory role of endogenous opioids on the immune system is not restricted to lymphocytes but can also take place at the level of cells belonging to T-cell differentiating microenvironments.     

Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity

DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly(644)-->Ser, Lys(643)Gly(644)-->TrpSer, or Asp(561)Lys(643)Gly(644)-->TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.     

Thymulin and the neuroendocrine system

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to this molecule. After its discovery in the early 1970, thymulin was characterized as a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, an emerging core of information points to thymulin as a hypophysotropic peptide. Here we review the evidence supporting the hypothesis that thymulin is an important player in the hypophyso-thymic axis.     

Contribution of the absolutely conserved B8Gly to the foldability of insulin

B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue. However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon. In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation. In order to identify more factors contributing to this positional conservation, the secretion efficiency, structural stability, disulfide stability, and in vitro refolding of single-chain insulin (PIP) and a mutant with B8Gly replaced by Ala, were investigated. Compared with wild-type PIP, the B8Ala replacement decreased the secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency of the PIP sequence. These results suggest that B8Gly is important to the secretion, folding, and stability of the insulin sequence.     

The inhibition of peptidyl transferase activity by aminoacyl derivatives of some nucleoside aliphatic analogues

Aminoacyl (Phe, Gly) derivatives of nucleoside aliphatic analogues bearing a hydroxyalkyl chain have been prepared by the condensation of the alcohols with N-benzyloxycarbonyl-amino acid in the presence of DCC followed by hydrogenolysis in methanol. These compounds inhibit peptidyl transferase activity and binding of acceptor substrate to E. coli ribosomes. The inhibitory activity is not much affected by the nature of either the aminoacyl or the heterocyclic base residue. In the transfer reaction, no peptide bond formation occurs with the above compounds as acceptors.     

青霉素G酰化酶β亚基Ser^579,Arg^580的定点突变研究

According to the comparison of amino acid sequence between PGA (Penicillin G Acylase) and PBPs (Penicillin Binding Protein), We suggest that No. 565-595 peptide fragment in beta-subunit of PGA may be a substrate-binding site of enzyme. Plasmid pTZGA was constructed by cloning the 2.6 kb PGA gene of pWGA into phagemid pTZ18U The technique of site-specific mutagenesis was used to study the role of residue No. 579 (Ser) and No. 580 (Arg) of PGA. Four kinds of mutants were obtained (Ser579-->Gly579, Arg580-->Gly580, Arg580-->Glu580, Arg580-->Lys580), both Glu580 and Gly580 mutants showed no activity of enzyme and Lys580 mutant remained 30% and Gly579 mutant kept 70% activity of wilde type. The same protein expression of four mutants according to the results of ELISA indicate that mutation does not affect the expression of PGA, but Arg580 residue may be essential for substrate-binding or catalysis of PGA.     

The glycine-rich sequence of the beta subunit of Escherichia coli H(+)-ATPase is important for activity

A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the beta subunit of Escherichia coli H(+)-ATPase (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151----Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H(+)-ATPase with these mutant beta subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the beta sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant beta subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.     

Biological activity and conformational isomerism in position 9 analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor from Saccharomyces cerevisiae

Analogues of the des-1-tryptophan,3-beta-cyclohexylalanine-alpha-factor of Saccharomyces cerevisiae, where the glycyl residue of position 9 was replaced by D-Ala, L-Ala, D-Leu, and L-Leu, were synthesized and evaluated by morphogenesis assays and circular dichroism spectroscopy. Synthesis was accomplished in solution phase with mixed anhydrides and p-nitrophenyl active esters as the coupling agents. All crude dodecapeptides were purified to greater than 98% homogeneity by preparative high-performance liquid chromatography on a reversed-phase column. The Gly9, D-Ala9, and D-Leu9 analogues elicited morphogenic alterations in MATa strains of S. cerevisiae at concentrations of 1-2 micrograms/mL and exhibited similar CD patterns in both trifluoroethanol and tris(hydroxymethyl)aminomethane buffer, pH 7.4. In contrast, the L-Ala9 and L-Leu9 analogues were more than 200 times less active in the morphogenesis assay and had markedly different CD spectra. These results demonstrate that the position 9 residue plays an important role in determining the biological activity and solution conformation of alpha-factor. We suggest the presence of a type II beta-turn in the Lys7-Gln10 region when the alpha-factor assumes its biologically active conformation.     

Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (approximately 10 microg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.     

Osteogenesis Imperfecta Model Peptides: Incorporation of Residues Replacing Gly within a Triple Helix Achieved by Renucleation and Local Flexibility

Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coefficients allowed the identification of partially folded species. When Gly was replaced by Ala, the Ala residue was incorporated into a fully folded triple helix, whereas replacement of Gly by Ser or Arg resulted in the presence of some partially folded species, suggesting a folding barrier. Increasing the triple-helix stability of the sequence N-terminal to a Gly-to-Ser replacement allowed complete triple-helix folding, whereas with the substitution of Arg, with its large side chain, the peptide achieved full folding only after flexible residues were introduced N-terminal to the mutation site. These studies shed light on the factors important for accommodation of Gly mutations within the triple helix and may relate to the varying severity of OI.