Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488146
Title: Investigation of the structure of fibrillin eight-cysteine motifs
Author: Dyer, Charlotte Emma
Awarding Body: University of Manchester : University of Manchester
Current Institution: University of Manchester
Date of Award: 1998
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Abstract:
The eight-cysteine motif is a protein repeat sequence that has been identified in extracellular matrix proteins of the fibrillin superfamily which includes isoforms fibrillin-l and fibrillin-2 and the closely related latent transforming growth factor-B binding proteins (LTBPs). These multidomain glycoproteins contain a number of cysteine-rich sequence repeats which resemble precursor epidermal growth factor domains (EGF-like domains) interspersed with eight-cysteine motifs and so-called "hybrid" domains, which exhibit some features of both these repeats. The signature of the eight-cysteine motif is a conserved pattern of eight"cysteine residues, three of which are contiguous. The structure of fibrillin eight-cysteine motifs has not been determined but mutations within fibrillin-l eight-cysteine motifs cause the heritable connective tissue disorder Marfan syndrome, confirming their critical role in fibrillin function and microfibril integrity. This study investigated the structure of fibrillin eight-cysteine motifs. Computer predictions of eight-cysteine motif secondary structure suggest limited amounts of defined secondary structure, most of which is beta-sheet (B-sheet), and a high proportion of loop regions. Several of the cysteine residues were predicted to be involved in disulphide bond formation. Eight-cysteine motif sequences did not fit to any known protein fold structures. Attempts to express a recombinant DNA molecule encoding a polyhistidine tagged human fibrillin-2 hybrid motif in mammalian and yeast cells, using plasmid expression vectors pCR™3 and pCS69 respectively, failed to produce detectable levels of recombinant protein, although a functional transcript and full-length polypeptide were produced using cell-free transcription and translation systems. However, a recombinant protein consisting of human fibrillin-l sixth eight-cysteine motif, flanked on either side by single EGF-like domains, was subsequently expressed using vector pSPEK in COS-I mammalian cells. This recombinant protein was N-glycosylated and exhibited anomalous migration during SDS-P AGE analysis, appearing larger than its predicted molecular weight of 18 kDa. Mass spectrometry analysis of the purified recombinant protein revealed products of masses 18 863 and 20 783 Da.. thought to correspond to non-glycosylated and glycosylated forms of. the protein respectively. Circular dichroism analysis of the recombinant protein revealed a high amount of ~-sheet structure (58% +/- 0.59), a small but significant alpha-helix (a-helix) content (1.0 +/- 0.56) and high amounts of residual structure (41 +/- 1.0), indicating significant amounts of ~-turn or loop regions. This study has provided insights into the secondary structure of eight-cysteine motifs. Computer predictions of secondary structure support data obtained by structural analysis of a recombinant protein containing a fibrillin eight-cysteine motif It is proposed that the eight-cysteine motif assumes a novel protein fold which contains a limited amount of defined secondary structure, most of which is ~-sheet with a small but significant amount of a-helix, the predominant feature of this motif being a high proportion of loop regions, which are held in place by disulphide bonding between pairs of the eight cysteine residues.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.488146  DOI: Not available
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