D A Kirschner — Page 6

PubMedDate not supplied

Designing recombinant spider silk proteins to control assembly.

The consensus repeat sequence found in the dragline silk from the spider, Nephila clavipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation. A synthetic gene incorporating the trigger was constructed, cloned and then expressed in Escherichia coli. The purified protein, about 25 kDa, contained the expected amino acid composition and migration behavior on SDS-PAGE. The recombinant protein was analyzed by X-ray diffraction, TEM, electron diffraction and circular dichroism in both oxidized and reduced states. Based on the results, the incorporation of a redox trigger appears to be a powerful experimental strategy to explore the self-assembly of fibrous proteins such as silks.

Amino Acid Sequence↗

PubMedDate not supplied

Generation of DM-20 splice site in myelin proteolipid protein gene: a hypothesis based on analysis of the amphibian protein.

The DM-20 isoform of proteolipid protein (PLP) of central nervous system myelin is undetectable in amphibia, but readily detectable in reptiles, birds and mammals. To explain the phylogenetic origin of DM-20, it has been proposed that a donor-acceptor RNA splice site was generated by the introduction of a single base change in the codon encoding amino acid 116 of PLP. We tested this hypothesis by isolating and sequencing peptides from bullfrog PLP. One of the peptides corresponded to mammalian residues 110-122, which contain the N-terminal boundary region for the domain excluded from DM-20 in higher vertebrates. We found that the Thr115-Val116 sequence is conserved between frog and mammal. Therefore, we propose that a single base change in the third position of the codon for Thr115 would account for the appearance of the new donor-acceptor splice site. Three amino acids elsewhere in this thirteen-residue peptide were found to differ between bullfrog and mammal, which could account for the previously reported weak recognition of amphibian PLP by an antiserum specific for peptide 109-128. A second peptide from bullfrog PLP had a sequence identical to that of residues 45-52 in mammalian PLP. Our findings explain how a specific change in the myelin PLP gene could generate a new form of the PLP protein.

Amino Acid Sequence↗