posted on 2023-11-07, 20:20authored byMirjam Hofmaier, Julian E. Heger, Sarah Lentz, Simona Schwarz, Peter Müller-Buschbaum, Thomas Scheibel, Andreas Fery, Martin Müller
Like multiblock copolymers,
spider silk proteins are built of repetitive
sequence motives. One prominent repetitive motif is based on the consensus
sequence of spidroin 4 of the spider Araneus diadematus ADF4. The number x of the repeating sequence motives
(C) determines the molecular weight of the recombinant ADF4-based,
engineered spider silk protein denoted as eADF4(Cx). eADF4(Cx) can be used as a model for intrinsically
disordered proteins (IDP) and to elucidate their folding. Herein,
the influence of the variation of the sequence motive repeating number x (x = 1, 2, 4, 8, 16) on the protein folding
within eADF4(Cx) films was investigated. eADF4(Cx)
films were cast from 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solutions
onto planar silicon model substrates, revealing mainly helical or
random coil structure. Upon treatment with methanol vapor (ptm), the
formation of crystalline β-sheets was triggered. Dichroic Fourier-transform
infrared (FTIR) spectroscopy, circular dichroism, spectroscopic ellipsometry,
atomic force microscopy, grazing-incidence small-angle X-ray scattering
(GISAXS), grazing-incidence wide-angle X-ray scattering (GIWAXS),
and electrokinetic and contact angle measurements were used to get
information concerning the secondary structure and folding kinetics,
orientation of β-sheets, the ratio of parallel/antiparallel
β-sheets, domain sizes and distributions, surface topography,
surface potential, hydrophobicity and the film integrity under water.
Significant differences in the final β-sheet content, the share
of antiparallel β-sheet structures, film integrity, surface
potential, and isoelectric points between eADF4(Cx) with x = 1, 2 and eADF4(Cx) with x = 4, 8, 16 gave new insights in the molecular weight-dependent
structure formation and film properties of IDP systems. GISAXS and
kinetic measurements confirmed a relation between β-sheet crystal
growth rate and final β-sheet crystal size. Further, competing
effects of reduced diffusibility hindering accelerated crystal growth
and enhanced backfolding promoting accelerated crystal growth with
increasing molecular weight were discussed.