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PubMed1972

Effects of proline and carbohydrates on the metabolism of exogenous proline by excised bean leaves in the dark.

Proline was metabolized when vacuum infiltrated into starved bean (Phaseolus vulgaris L.) leaves from plants previously in the dark for 48 hours, but an equivalent increase in protein proline was not observed. When (14)C-proline was infiltrated into starved leaves, a large percentage of the (14)C was recovered in other amino acids, organic acids, and CO(2), in addition to that recovered as protein proline. However, extensive oxidation of proline was observed only if enough proline was added to increase substantially the endogenous concentration of proline. Increasing the endogenous concentration did not affect the amount of proline that was incorporated into protein.When added to leaves from plants previously in the light of near saturating intensity for more than 16 hr, very little (14)C-proline was oxidized, even when a sufficient amount of proline was added to increase the endogenous concentration. Adding sucrose to starved leaves along with the proline slowed down the oxidation of proline. Thus, it appears that some carbohydrate or intermediate of carbohydrate metabolism may inhibit the oxidation of proline in leaves.Based on kinetics of labeling, the pathway of proline oxidation was by conversion to glutamic acid and subsequent metabolism to intermediates in the Krebs cycle and to CO(2).

Journal Article↗

PubMed1978

Metabolism of [5-h]proline by barley leaves and its use in measuring the effects of water stress on proline oxidation.

The objective of these experiments was to determine the fate of tritium from the 5 position of proline and to assess the validity of its loss to H(2)O as a measure of proline oxidation. When [5-(3)H]proline was fed to barley (Hordeum vulgare) leaves, tritium was recovered in H(2)O and metabolites such as glutamate, glutamine, organic acids, aspartate, asparagine, and gamma-aminobutyrate. Collectively these metabolites, which are oxidation products of proline, accounted for 8% of the (3)H recovered after 5 hours. In spite of the amount recovered in metabolites, the rates of proline oxidation estimated by measuring (3)H(2)O recovery from [5-(3)H]proline were only slightly lower than rates estimated by incorporation of (14)C into oxidized products and loss of (14)C from total proline. Therefore, (3)H(2)O recovery from [5-(3)H]proline is useful in assessing the effects of stress on proline metabolism.Water stress inhibited proline oxidation, as reported previously. In addition, a reconversion of proline oxidation products to proline occurred in stressed leaves. This observation probably indicates a breakdown in cellular compartmentation of proline synthesis and proline oxidation.

Journal Article↗

PubMed1984

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism.

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0(2) uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited big up tri, open(1)-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.The effect of T4C on proline metabolism in detached green barley leaves was investigated. T4C inhibited proline oxidation in turgid leaves, increasing the proline content of these leaves slightly. In wilted leaves (that are synthesizing proline rapidly), T4C inhibited proline synthesis, which resulted in a decrease in the proline content of the leaves. big up tri, open(1)-pyrrolidine-5-carboxylic acid reductase (the last enzyme in proline synthesis) was not inhibited by T4C, and thus T4C's influence is prior to that step of the synthetic pathway. T4C had no influence on the incorporation of proline into protein.

Journal Article↗

PubMed1995

Proline dehydrogenase activity of the transcriptional repressor PutA is required for induction of the put operon by proline.

The proline utilization (put) operon from Salmonella typhimurium consists of the putP gene, encoding a proline transporter, and the putA gene, encoding an enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities. In addition to these two enzymatic activities, the PutA protein is a transcriptional repressor that regulates the expression of putP and putA in response to the availability of proline. We report the isolation of super-repressor mutants of PutA that decrease expression from the putA promoter in the presence or absence of proline. None of the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro. Although DNA binding by wild-type PutA was prevented by the addition of proline and an artificial electron acceptor, DNA binding by the two strongest super-repressors was not prevented under identical conditions. The proline dehydrogenase activity of the purified mutant proteins showed altered kinetic properties (increased Km(Pro), reduced Vmax, or a completely null phenotype). The observation that these mutations simultaneously affect induction by proline and proline dehydrogenase activity suggests that a single proline-binding site is involved in both proline dehydrogenase activity and induction of the expression of the put operon. Furthermore, the results indicate that the proline dehydrogenase activity of PutA is essential for induction of the put operon by proline.

Alleles↗

PubMed1986

The effects of benzyladenine, cycloheximide, and cordycepin on wilting-induced abscisic Acid and proline accumulations and abscisic Acid- and salt-induced proline accumulation in barley leaves.

Benzyladenine inhibits proline accumulation in wilted, abscisic acid (ABA)-treated, and salt-shocked barley leaves. It does not affect ABA accumulation or disappearance in wilted leaves. Inhibition of proline accumulation in salt-shocked leaves was observed both when benzyladenine was added at the beginning of or after salt treatment. Cycloheximide (CHX) and cordycepin inhibited both ABA and proline accumulations in wilted barley leaves and proline accumulation in ABA-treated leaves. In salt-shocked leaves, cordycepin inhibited proline accumulation when added after salt treatment but before proline began to accumulate but not when added after the onset of proline accumulation. CHX delayed the accumulation of proline in salt-shocked leaves but, after a period of time, proline accumulated in the CHX-treated leaves at rates comparable to the salt-treated control. This delay and subsequent accumulation was observed when CHX was added before, during, and after salt treatment. However, the earlier in the salt treatment period that CHX was given, the longer was the observed delay. These results are interpreted to indicate that gene activation is involved in proline accumulation in response to wilting, to ABA, and to salt in barley leaves. This gene activation is in addition to the gene activation that is required for ABA accumulation in wilted leaves. If ABA accumulation is required for proline accumulation in wilted barley leaves, then two sets of gene activation are involved in wilting-induced proline accumulation. All of our results are consistent with this possibility but do not prove it. The inhibition of proline accumulation by benzyladenine is probably neither due to an effect on gene activation nor to an effect on the ABA level.

Journal Article↗

PubMed1979

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.

Enzyme Induction↗

PubMed1999

Stimulation of benzyladenine-induced in vitro shoot organogenesis and endogenous proline in melon (Cucumis melo L.) by fish protein hydrolysates in combination with proline analogues.

A previous study demonstrated that proline is beneficial for improving melon in vitro shoot organogenesis. A natural source of proline and proline precursors can be obtained from fish protein hydrolysates (FPH), a byproduct of the fishery industry. Proline analogues azetidine-2-carboxylate and hydroxyproline in combination with standardized FPH were used to stimulate proline synthesis and benzyladenine-induced shoot organogenesis by exploiting the proposed proline-linked pentose phosphate pathway (PPP). In the presence of elevated levels of endogenous proline, potential stimulation of cytokinins and auxin may occur via the PPP and shikimate pathways, respectively. Treatments with FPH singly and in combination with the above proline analogues significantly increased the endogenous proline content and the extent of differentiation, suggesting that in vitro organogenesis is closely linked to proline synthesis, strengthening the hypothesis that purine metabolism via the proline-linked PPP may be important for organogenesis. Thioproline addition resulted in increased proline levels but without corresponding stimulation of organogenesis. This study also provides potential use of fishery waste for value-added application in plant micropropagation industry.

Adenine↗

PubMed1972

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium.

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.

Catalysis↗

PubMed1996

Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants.

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1989

Effect of dietary proline on proline metabolism in the neonatal pig.

The effect of two dietary concentrations of proline (10.3 and 15.8 g/kg) on proline-metabolizing enzymes [pyrroline-5-carboxylate (P5C) reductase and proline oxidase], plasma and tissue free proline concentrations and growth were investigated in the 2- to 13-d-old pig. Diet had no effect on growth or enzyme activity. Diet had a significant (P less than 0.05) effect on the concentration of free proline in plasma, liver, intestine and muscle, but no effect in kidney. These data suggest that the magnitude and pattern of change of P5C reductase activity is not influenced by the concentration of proline in the diet. The lower plasma and tissue free proline concentrations in the piglets fed the basal diet compared with piglets fed the proline-supplemented diet and the lack of effect of diet on enzyme activity suggest there was inadequate proline in the basal diet, and those piglets were unable to increase proline synthesis to maintain normal proline concentrations.

Age Factors↗

PubMed1989

Proline and proline derivatives as anticonvulsants.

1. The anticonvulsant properties of L-proline, of proline derivatives (trans-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, 3,4-dehydro-D,L-proline) and of D- and L-pipecolic acid were studied alone and in combination with vigabatrin (R/S-4-aminohex-5-enoic acid). 3-Mercaptopropionic acid and pentylenetetrazol-induced convulsions in mice were used as animal models of epilepsy. 2. Proline and proline derivatives are weak anticonvulsants if given alone in doses up to 10 mmol/kg, however, they are capable of potentiating the anticonvulsant effects of vigabatrin, in a manner similar to that reported previously for glycine, and some glycine derivatives. Among the compounds tested, trans-4-hydroxy-L-proline was the most potent anticonvulsant in combination with the indirect GABA agonist vigabatrin. 3. A potential explanation for the synergistic anticonvulsant effect of the combination of the GABA agonist and proline is the presumed role of proline as inhibitory neurotransmitter, and/or its glutamate antagonistic effects. 4. The current study points out the lack of basic knowledge on the neurochemistry and pharmacology of proline and hydroxyproline.

3-Mercaptopropionic Acid↗

PubMed2003

Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae.

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.

Freezing↗

PubMed1983

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro.

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.

Aminopeptidases↗

PubMed1996

Detection of Novel Proline 3-Hydroxylase Activities in Streptomyces and Bacillus spp. by Regio- and Stereospecific Hydroxylation of l-Proline.

During the screening of microbial proline hydroxylases, novel proline 3-hydroxylase activities, which hydroxylate free l-proline to free cis-3-hydroxy-l-proline, were detected in whole cells of Streptomyces sp. strain TH1 and Bacillus sp. strains TH2 and TH3 from 3,000 strains isolated from soil. The reaction product was purified from a reaction mixture of Streptomyces sp. strain TH1, and its chemical structure was identified as cis-3-hydroxy-l-proline by instrumental analyses. Proline 3-hydroxylase activity was also detected in Streptomyces canus ATCC 12647 which produces the 3-hydroxyproline-containing peptide antibiotic telomycin. Bacillus sp. strains TH2 and TH3 were found to accumulate cis-3-hydroxy-l-proline in culture media at 426 and 352 (mu)M, respectively. It was suggested that hydroxylation occurred in a highly regio- and stereospecific manner at position 3 of l-proline because no hydroxylation product other than cis-3-hydroxy-l-proline was observed. Proline 3-hydroxylases of these strains were first characterized on crude enzyme preparations. Since 2-oxoglutarate and ferrous ion were required for hydroxylation of l-proline, these 3-hydroxylases were thought to belong to a family of 2-oxoglutarate-related dioxygenases. The reaction was inhibited by Co(sup2+), Zn(sup2+), and Cu(sup2+). l-Ascorbic acid accelerated the reaction. The optimum pH and temperature were 7.5 and 35(deg)C, respectively.

Journal Article↗

PubMed2000

Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline.

A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.

Amino Acid Sequence↗

PubMed1987

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures.

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.

Amino Acids↗

PubMed1982

Nitrogen fixation in Klebsiella pneumoniae during osmotic stress. Effect of exogenous proline or a proline overproducing plasmid.

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity of Klebsiella pneumoniae, wild-type strain M5A1. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F' conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixation by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.

Klebsiella pneumoniae↗

PubMed1991

Cell adhesion promoting peptide GVKGDKGNPGWPGAP from the collagen type IV triple helix: cis/trans proline-induced multiple 1H NMR conformations and evidence for a KG/PG multiple turn repeat motif in the all-trans proline state.

Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by 1H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. At least five such states are observed, for example, for the well-resolved A14 beta H3 group, and K3, which is six residues sequentially removed from the nearest proline, i.e., P9, shows two sets. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 and A14-trans-P15, and more than one set for a residue sequentially well-removed from a proline, e.g., K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D 1H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. Peptide IV-H1 in the all-trans proline state ttt exists in relatively well-defined conformation populations showing numerous short- and long-range NOEs and long-lived backbone amide protons and reduced backbone NH temperature coefficients, suggesting hydrogen-bonding, and structurally informative 3J alpha N coupling constants. The NMR data indicate significant beta-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal gamma-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% beta-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein. Synthetic peptide IV-H1 overlapping sequence "walk throughs" indicate that the primary biological activity is localized in the GNPGWPGAP double beta-turn domain, which contains the backbone constraining proline residues. This proline-domain conformation may suggest a collagen type IV receptor-specific, metastatic cell adhesion promoting binding domain.

Amino Acid Sequence↗