High molecular mass complexes of aquatic silk proteins

Abstract

Little is known about specific protein–protein associations that take place during formation of Chironomus tentans silk. The aim of this study was to learn if C. tentans salivary glands contain biochemically discrete silk protein complexes. Examination of native extracts by non-denaturing agarose gel electrophoresis and immunoblotting revealed two SDS-resistant complexes: C1a, nominally containing silk proteins spIa, sp185 and sp140, and C1b, containing spIb, sp185 and sp140. The data also implied that C1a and C1b can further associate into SDS-sensitive homo- or hetero-oligomers. Sedimentation of extracts in preparative glycerol gradients resulted in a heterogeneous distribution of C1a and C1b centered near 30S. Examination of gradient fractions by denaturing polyacrylamide gel electrophoresis and immunoblotting indicated that C1a and C1b co-sediment with spIs, sp185, and sp140; however, these fractions also contained sp40, sp17 and sp12. In contrast, two other silk proteins sedimented throughout the gradient. Electron micrographs of a complex-containing fraction showed discrete, sometimes oligomeric lattice-like structures that, over time, assembled in vitro into multistranded beaded fibers. It is proposed that C1a and C1b are quaternary structures that are intermediates in the assembly pathway of C. tentans silk.