Protein fiber extensibility and strength: GGX and space motif contributions to mechanical and structural properties of flagelliform spider silk determined through recombinant protein characterization

Abstract

Flagelliform spider silk is a highly extensible protein polymer produced by orb weaver spiders with remarkable mechanical properties combining strength with incredible extensibility. The flagelliform fibers are composed of a single protein, Flag, and can stretch up to six times their original length. The core of the large 400 kDa Flag protein is composed of three simple amino acid motifs. The GPGGX motif has been previously shown to contribute the elasticity of the silk fibers but the source of the strength of these fibers has been elusive. The GGX and “spacer” core motifs were selected for the purpose of this study to determine their specific contributions to the fiber mechanical properties and structure. Four recombinant proteins were designed and genetically engineered to contain repeats of the GGX; the GGX and GPGGX, the GGX and spacer; or the GGX, spacer and GPGGX modules. The recombinant proteins, ranging in size from 55-65 kDa, were utilized as molecular tools to investigate the mechanical property contributions of the individual motifs to the silk fibers. Structural studies of the recombinant silk fibers provided new insight into motif structural contributions of the native flagelliform Flag protein. Raman spectroscopic analyses correlate with an increase in β-turn structure with increased spacer motifs in the recombinant proteins. Wide Angle X-ray diffraction studies provide evidence of increased molecular structural orientation correlating with the inclusion of the spacer motif. The GGX and GPGGX fibers produce amorphous X-ray diffraction data. These results suggest that the spacer motif is a major contributor to the strength of native flagelliform silk. These recombinant proteins provide a platform for additional studies that will contribute to the understanding of the structure/function correlation that produce the material mechanical properties of flagelliform silk.