Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n >= 2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)(8). All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-beta structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-beta-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into beta-sheet structures, which is necessary for nucleation and beneficial for fibril growth. (C) 2014 Elsevier Inc. All rights reserved.