Reduction-oxidation control of beta-sheet assembly in genetically engineered silk

Genetically engineered spider dragline silk protein was modified to incorpo rate methionines flanking the beta -sheet forming polyalanine regions. The methionines could be selectively chemically oxidized and reduced. This chem ical change altered the bulkiness and charge of the sulfhydryl groups, and in turn, the beta -sheet forming tendencies of the polyalanine domains and solubility of the protein. The genes encoding these redesigned proteins wer e constructed, cloned and expressed in Escherichia coli. In the reduced sta te (beta -mercaptoethanol) the similar to 25 kDa protein behaved similarly to native spider dragline silk, crystallizing into beta -sheets based on di ffraction analysis and appearing fibrous by TEM. The addition of the methio nines into the consensus dragline silk sequence did not disrupt the normal macromolecular assembly behavior of the protein. In the oxidized state (phe nacyl bromide) the protein did not form beta -sheet crystals and appeared m orphologically featureless based on TEM. A reduction in beta -strand conten t was also observed upon oxidation based on FTIR and TEM analysis and confi rmed by X-ray diffraction analysis. To further confirm changes in assembly behavior observed for the recombinant protein containing the methionines, a model peptide with the same repeat amino acid sequence was synthesized and characterized. Shifts in molecular weight, observed by MALDI, along with c orresponding changes in crystallinity, by electron diffraction, agreed with the changes expected on activation and deactivation of the redox trigger. These results support the use of a redox trigger as a useful feature with w hich to control the assembly of beta -sheet forming proteins.