Triggered assembly of spider-silk like proteins

While recombinant spider-silk proteins have been successfully generated, a significant limitation has been the difficulty in maintaining solubility of these proteins. Premature precipitation arises due to crystallization of the protein into β-sheet domains, rendering it insoluble in aqueous and buffered systems. The control of β-sheet formation is thus an important factor in the understanding and prediction of structural transitions and protein folding. In this work, control of this process has been achieved by genetically engineering spider-silk proteins to incorporate sterical triggers.; Spider-silk protein, based on the consensus sequence of Nephila clavipes dragline-silk, was genetically engineered to include methionines flanking the β-sheet forming poly-alanine regions. These methionines could be selectively reduced and oxidized, altering the bulkiness and charge of the methionine sulfhydryl group to control beta-sheet formation by steric hindrance. A second version of the sterical trigger included a recognition site for Protein-Kinase-A allowing for the selective phosphorylation of a serine. The recombinant silk genes were constructed using a bacterial vector shuttle system and were expressed in Escherichia coli.; The silk proteins were analyzed using circular-dichroism, transmission-electron-microscopy and X-ray diffraction to determine if the inclusion of the trigger sequences would interfere with the formation of the characteristic β-sheet structures of silk like proteins. The proper function and reversibility of the sterical triggers was also shown using MALDI on model peptides based on the monomeric repeat of the silk proteins.; Structural trigger assessment was achieved through biophysical characterization and monitoring structural transitions by attenuated-total-reflectance-infrared-spectroscopy for solution state structures in both triggered and untriggered forms. For solid state structural characterization, IR-microscopy, reflectance-IR, electron-diffraction data, transmission-electron-microscopy and circular-dichroism studies provided structural corroboration for all experiments.; The results indicate that the intractable β-sheet formation of silk-like proteins can be influenced by the use of sterical trigger designs. This process may be a useful model to consider in studies of β-sheet assembly in general, including β-amyloid and prion systems. An understanding of the folding and assembly pathways for silks, as well as for these other groups of proteins with similar secondary structures, may provide opportunities to disrupt these processes in cases where this is desirable.