Dichroic Fourier Transform Infrared Spectroscopy Characterization of the β-Sheet Orientation in Spider Silk Films on Silicon Substrates

Abstract

Orientation analysis of the β-sheet structure within films of the established recombinant spider silk protein eADF4­(C16) was performed using a concept based on dichroic transmission– and attenuated total reflection–Fourier transform infrared spectroscopy, lineshape analysis, assignment of amide I components to specific vibration modes, and transition dipole moment directions of β-sheet structures. Based on the experimental dichroic ratio R, the order parameter S of β-sheet structures was calculated with respect to uniaxial orientation. Films of eADF4­(C16) were deposited on untexturized (Si) and unidirectionally scratched silicon substrates (Si-sc) and post-treated with MeOH vapor. Freshly cast thin and thick eADF4­(C16) films out of hexafluoroisopropanol featured β-sheet contents of ≈6%, which increased to >30% after MeOH post-treatment in dependence of time. Pseudo-first order folding kinetics were obtained, suggesting a transition from an unfolded to a folded state. In MeOH post-treated thin films with diameters in the nanometer range, a significant orientation of β-sheets was obtained regardless of the texturization of the silicon substrate (Si, Si-sc). This was rationalized by dichroic ratios of the amide I component at 1696 cm–1 assigned to the (0, π) mode of antiparallel β-sheet structures, whose transition dipole moment M is located in parallel to both β-sheet plane and chain direction. The calculated high molecular order parameter S ≈ 0.40 suggested vertically (out-of-plane) oriented antiparallel β-sheet stacks with tilt angles of γ ≈ 39° to the surface normal. Microscale (thick) films, in contrast, revealed low order parameters S ≈ 0. Scanning force microscopy on thin eADF4 films at silicon substrates showed dewetted polymer film structures rather at the micro-scale. These findings give new insights in the role of the β-sheet crystallite orientation for the mechanical properties of spider silk materials.

β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides

“…As published previously, ,, the weaker band component at 1697 cm –1 visible in Figure B,D is assigned exclusively to the (0, π) mode of the antiparallel β-sheet. The angle θ th between the transition dipole moment M and both the plane and polypeptide chain direction in the elementary unit of this mode is 0° (“parallel”, “ in-plane ” mode, see SI, Figure S3).…”

“…The thickness of spider silk protein films can be carefully adjusted by the concentration of the spider silk solution as shown previously. , Further, the film thickness highly influences the self-assembly behavior in protein films. Crystalline β-sheets are oriented toward the protein/air and the substrate/protein interface and forming micelle-like inclusions embedded in the amorphous matrix in microfilms. , Moreover, the dependence of thickness and adsorption in the ATR-FTIR method used here (see Secondary Structure of pep-c/pep-a Blend Films) is only quasilinear in a range from d = 0 nm to d = 300 nm film thickness .…”

“…Exemplary dichroic ATR-FTIR spectra of blend-50 (A, B) and eADF4(C1) (C, D) films as-cast (A, C) and after ptm (B, D). Dichroic ATR-FTIR spectra of cast eADF4(C16) films with comparable thickness have been published earlier 33. P-polarized spectra are depicted in black and s-polarized spectra are depicted in red.…”

“…33 Using the respective dichroic ratios R = B P /B S obtained by dichroic TRANS-FTIR and ATR-FTIR spectroscopy, the orientation of all films was analyzed using the Amide I band intensities according to Miyazawa, Blout, and Krimm. 44,47,60 As published previously, 33,60,77 the weaker band component at 1697 cm −1 visible in Figure 6B,D is assigned exclusively to the (0, π) mode of the antiparallel β-sheet. The angle θ th between the transition dipole moment M and both the plane and polypeptide chain direction in the elementary unit of this mode is 0°("parallel", "in-plane" mode, see SI, Figure S3).…”

“…Note that previously published S z values of eADF4(C16) were lower due to different film preparation and parameters used for LSA. 33 It is assumed that the mentioned chemical connectivity between crystalline and amorphous regions in eADF4(C1) and All blend and pep-c films showed comparable and the highest order parameters indicating the best orientation toward the z-axis. For both eADF4(C1) and eADF4(C16) films lower but relevant order parameters comparable to pep-a films after ptm were obtained.…”