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1.
Protein Sci ; 10(4): 735-40, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11274464

ABSTRACT

Tyrosine residues of neuroendocrine peptides are frequently the targets of oxidation reactions, one of which involves hydroxylation to peptidyl-3, 4-dihydroxy-phenyl-L-alanine (DOPA). The reactivity in vitro of peptidyl-DOPA in two neuroendocrine peptides, a neurotensin fragment (pELYENK) and proctolin (RYLPT), was investigated using ultraviolet-visible scanning spectrophotometry and matrix-assisted laser desorption ionization mass spectrometry following oxidation by tyrosinase and periodate. The peptides form covalently coupled dimers and trimers, and their masses are consistent with the presence of diDOPA cross-links. Lysine does not appear to participate in multimer formation because it is efficiently recovered in fragmentation ladders using subtilisin. While multimer formation in the neurotensin-derived peptide can be blocked effectively by adding N-acetyl-DOPA-ethylester to the reaction medium, the DOPA ethylester couples itself four to five times to each peptide.


Subject(s)
Dihydroxyphenylalanine/analogs & derivatives , Monophenol Monooxygenase/chemistry , Neuropeptides/chemistry , Neurotensin/chemistry , Oligopeptides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tyrosine/chemistry , Agaricales/enzymology , Cross-Linking Reagents/chemistry , Humans , Hydroxylation , Oxidation-Reduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Spectrophotometry/instrumentation , Spectrophotometry/methods
2.
Biochemistry ; 39(36): 11147-53, 2000 Sep 12.
Article in English | MEDLINE | ID: mdl-10998254

ABSTRACT

Mytilus edulis foot protein-1 (mefp1) is a major component of the byssus, an adhesive holdfast in mussels. The recent report of 5, 5'-di(dihydroxyphenyl-L-alanine) (diDOPA) cross-links in byssus [McDowell et al. (1999) J. Biol. Chem. 274, 20293] has raised questions about the relationship of these to mefp1. About 80% of the primary structure of mefp1 consists of a tandemly repeated consensus sequence Ala(1)-Lys(2)-Pro(3)-Ser(4)-Tyr(5)-Pro(6)-Pro(7)-Thr(8)-Tyr(9)-Lys(10 ) with varying degrees of posttranslational hydroxylation to hydroxyprolines in positions 3, 6, and 7 and to DOPA in positions 5 and 9. Six natural or synthetic variants of this decapeptide were subjected to oxidation by tyrosinase or periodate. DOPA is the only residue to suffer losses in all oxidized peptides. Moreover, using MALDI TOF mass spectrometry, oxidized decapeptides all showed evidence of multimer formation and a mass loss of 6 Da per coupled pair of peptides. Multimer formation was inhibited by addition of DOPA-like o-diphenols, but addition of simple amines such as free Lys had no effect. The results are consistent with aryloxy coupling to diDOPA followed by reoxidation to diDOPA quinone. There are subtle but noteworthy variations, however, in multimer formation among the peptide congeners. Decapeptides with Pro(3) modified to trans-4-hydroxyproline do not form multimers beyond dimers; they also exhibit significant Lys losses following oxidation of DOPA. Moreover, in Ala-Lys-Hyp-Ser-Tyr-DiHyp-Hyp-Thr-DOPA-Lys, Tyr appears to be protected from oxidation by tyrosinase.


Subject(s)
Adhesives/chemistry , Cross-Linking Reagents/chemistry , Models, Molecular , Oligopeptides/chemistry , Proteins/chemistry , Adhesiveness , Adhesives/metabolism , Animals , Bivalvia , Cross-Linking Reagents/metabolism , Dihydroxyphenylalanine/chemistry , Dihydroxyphenylalanine/metabolism , Glycine/metabolism , Hydrolysis , Lysine/metabolism , Oligopeptides/metabolism , Oxidation-Reduction , Proteins/metabolism , Quinones/chemistry , Quinones/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, Ultraviolet , Tyrosine/metabolism
3.
Biochim Biophys Acta ; 1479(1-2): 315-20, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-11004549

ABSTRACT

The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure.


Subject(s)
Glycine/chemistry , Proline/chemistry , Proteins/metabolism , Amino Acid Sequence , Animals , Bivalvia , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Peptide Mapping , Proteins/chemistry , Proteins/isolation & purification , Species Specificity , Trypsin/chemistry
4.
Article in English | MEDLINE | ID: mdl-11007180

ABSTRACT

The in vitro enzymatic polymerization of the polyphenolic protein purified from the mussels Aulacomya ater, Mytilus edulis chilensis and Choromytilus chorus was studied. Mushroom tyrosinase was used to oxidize the dopa residues present in these proteins, and polymerization was monitored by acid-urea polyacrylamide gel electrophoresis. The protein from A. ater polymerized at a faster rate than the other two. Amino acid analysis of the crosslinked protein showed a notable decrease in the content of dopa, but no significant change of other amino acids. This suggests that crosslink formation may be limited to the oxidized dopa derivatives of the protein molecules.


Subject(s)
Agaricales/enzymology , Bivalvia/metabolism , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/metabolism , Proteins/chemistry , Proteins/metabolism , Animals , Cross-Linking Reagents/metabolism , Dimerization , Substrate Specificity
5.
J Biol Chem ; 274(29): 20293-5, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10400649

ABSTRACT

13C2H rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13C]tyrosine and L-[ring-d4]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5'-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.


Subject(s)
Bivalvia/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Animals , Cross-Linking Reagents/chemistry , Tyrosine/chemistry
6.
Curr Opin Biotechnol ; 8(3): 309-12, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9206011

ABSTRACT

Many marine organisms attach to underwater surfaces using protein adhesives. These are basic proteins with high levels of the amino acid 3,4-dihydroxyphenylalanine and an extended flexible conformation. The hydroxylation of tyrosine residues plays a key role in the chemisorption of these polymers to surfaces and in the setting of the adhesive. These unique proteins are attracting biotechnological attention for application in industry and medicine. Recent development on the immobilization of antigens and antibodies, enzymes, cells and tissues, illustrate the great potential use of these adhesives for diagnostics and medicine. The use of these adhesive proteins as anticorrosive coats for metal also suggests important applications for industry.


Subject(s)
Adhesives/isolation & purification , Marine Biology , Proteins/isolation & purification , Adhesiveness , Amino Acid Sequence , Animals , Biotechnology , Bivalvia/chemistry , Bivalvia/genetics , Consensus Sequence , Proteins/genetics
7.
Arch Biochem Biophys ; 333(1): 221-4, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8806774

ABSTRACT

Rotational-echo double-resonance (REDOR) 13C NMR spectra with 2H dephasing have been obtained from plaques and threads from the byssus of the marine mussel Mytilus edulis labeled by sea-water exposure to L-[ring-4-(13)C]tyrosine and L-[ring-d4]tyrosine for 2 days. Specific isotopic enrichment of tyrosine in protein reached 25% in both 13C and 2H. Fifteen percent of the total 13C label was incorporated as diphenolic carbon. Based on REDOR dephasing, about one-tenth of tyrosine rings in both intact plaques and threads are within 4 A of each other or rings of 3,4-dihydroxy-phenylalanine (DOPA). However, there is no direct evidence for the formation of covalent linkages between or among tyrosine and DOPA rings in either plaques or threads.


Subject(s)
Bivalvia/chemistry , Dihydroxyphenylalanine/analogs & derivatives , Dihydroxyphenylalanine/chemistry , Peptides/chemistry , Animals , Carbon Isotopes , Cross-Linking Reagents , Deuterium , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy/methods , Oxidation-Reduction , Tyrosine/chemistry
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