Influence of the Sequence Motive Repeating Number on Protein Folding in Spider Silk Protein Films
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Contributors
Abstract
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.
Details
Original language | English |
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Pages (from-to) | 5707-5721 |
Number of pages | 15 |
Journal | Biomacromolecules |
Volume | 24 |
Issue number | 12 |
Early online date | 7 Nov 2023 |
Publication status | Published - 11 Dec 2023 |
Peer-reviewed | Yes |
External IDs
PubMed | 37934893 |
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