Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins

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Humenik, Martin ; Magdeburg, Michael ; Scheibel, Thomas:
Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins.
In: Journal of Structural Biology. Bd. 186 (2014) Heft 3 . - S. 431-437.
ISSN 1047-8477
DOI: https://doi.org/10.1016/j.jsb.2014.03.010

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Abstract

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 nP2. 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-b 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-b-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 b-sheet structures, which is necessary for nucleation and
beneficial for fibril growth