title: Reduction-oxidation control of beta-sheet assembly in genetically engineered silk.
abstract: Genetically engineered spider dragline silk protein was modified to incorporate methionines flanking the beta-sheet forming polyalanine regions. The methionines could be selectively chemically oxidized and reduced. This chemical 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 were constructed, cloned and expressed in Escherichia coli. In the reduced state (beta-mercaptoethanol) the approximately 25 kDa protein behaved similarly to native spider dragline silk, crystallizing into beta-sheets based on diffraction analysis and appearing fibrous by TEM. The addition of the methionines into the consensus dragline silk sequence did not disrupt the normal macromolecular assembly behavior of the protein. In the oxidized state (phenacyl bromide) the protein did not form beta-sheet crystals and appeared morphologically featureless based on TEM. A reduction in beta-strand content was also observed upon oxidation based on FTIR and TEM analysis and confirmed 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 corresponding 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 which to control the assembly of beta-sheet forming proteins.
authors.affiliation: Department of Chemical Engineering, Biotechnology Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, USA.
authors.forename: S
authors.initials: S
authors.lastname: Szela
authors.forename: P
authors.initials: P
authors.lastname: Avtges
authors.forename: R
authors.initials: R
authors.lastname: Valluzzi
authors.forename: S
authors.initials: S
authors.lastname: Winkler
authors.forename: D
authors.initials: D
authors.lastname: Wilson
authors.forename: D
authors.initials: D
authors.lastname: Kirschner
authors.forename: D L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013112:Spiders / Q000235:genetics*
chemical_list: D000596:Amino Acids; D011506:Proteins