Swelling and Orientation Behavior of End-Grafted Polymer Chains by In Situ Attenuated Total Reflection Fourier Transform Infrared Spectroscopy Complementing In Situ Ellipsometry.

The literature lacks established concrete parameters for assigning grafted chain regimes. In this context, dichroic in situ attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and in situ ellipsometry were used complementarily, offering new opportunities for conformational analysis of end-grafted polymer chains. Especially polymer chain orientation was studied as a new parameter, among others, for proper chain regime assignment in this report. Alkyne-functionalized poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with a molecular weight of 49.8 kg/mol and a contour length of around 80 nm was grafted to self-assembled monolayers bearing triazole end groups as reported. Different chain regimes were generated by using three different grafting densities. ATR-FTIR spectroscopy based on the ν(C═O) stretching vibration at around 1728 cm-1 provided a new direct approach to determine the GD of polymer chains. Significant shifts in the position of the ν(C═O) band comparing dry and wet states were observed, caused by increased hydrogen bonding interactions between PDMAEMA and water. Finally, the averaged orientation of PDMAEMA chains along the z-axis was determined using dichroic ATR-FTIR spectroscopy based on the dichroic ratios of the ν(C═O) band and molecular order parameters SZ,MOL calculated thereof. High SZ,MOL values were found for the wet state compared to the dry state, confirming that all GD PDMAEMA samples are in the brush regime in the swollen state.

Influence of the Sequence Motive Repeating Number on Protein Folding in Spider Silk Protein Films.

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 ß-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 ß-sheets, the ratio of parallel/antiparallel ß-sheets, domain sizes and distributions, surface topography, surface potential, hydrophobicity and the film integrity under water. Significant differences in the final ß-sheet content, the share of antiparallel ß-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 ß-sheet crystal growth rate and final ß-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.

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

Intrinsically disordered proteins (IDPs) play an important role in molecular biology and medicine because their induced folding can lead to so-called conformational diseases, where ß-amyloids play an important role. Still, the molecular folding process into the different substructures, such as parallel/antiparallel or extended ß-sheet/crossed ß-sheet is not fully understood. The recombinant spider silk protein eADF4(Cx) consisting of repeating modules C, which are composed of a crystalline (pep-c) and an amorphous peptide sequence (pep-a), can be used as a model system for IDP since it can assemble into similar structures. In this work, blend films of the pep-c and pep-a sequences were investigated to modulate the ß-sheet formation by varying the molar fraction of pep-c and pep-a. Dichroic Fourier-transform infrared spectroscopy (FTIR), circular dichroism, spectroscopic ellipsometry, atomic force microscopy, and IR nanospectroscopy were used to examine the secondary structure, the formation of parallel and antiparallel ß-sheets, their orientation, and the microscopic roughness and phase formation within peptide blend films upon methanol post-treatment. New insights into the formation of filament-like structures in these silk blend films were obtained. Filament-like structures could be locally assigned to ß-sheet-rich structures. Further, the antiparallel or parallel character and the orientation of the formed ß-sheets could be clearly determined. Finally, the ideal ratio of pep-a and pep-c sequences found in the fibroin 4 of the major ampullate silk of spiders could also be rationalized by comparing the blend and spider silk protein systems.

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

Orientation analysis of the ß-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 ß-sheet structures. Based on the experimental dichroic ratio R, the order parameter S of ß-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 ß-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 ß-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 ß-sheet structures, whose transition dipole moment M is located in parallel to both ß-sheet plane and chain direction. The calculated high molecular order parameter S ≈ 0.40 suggested vertically (out-of-plane) oriented antiparallel ß-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 ß-sheet crystallite orientation for the mechanical properties of spider silk materials.

Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with siRNA.

In the context of gene delivery, chitosan has been widely used as a safe and effective polycation to complex DNA, RNA and more recently, siRNA. However, much less attention has been paid to chitosan oligosaccharides (COS) despite their biological properties. This study proposed to carry out a physicochemical study of COS varying in degree of polymerization (DP) from 5 to 50, both from the point of view of the solution properties and the complexing behavior with siRNA. The main parameters studied as a function of DP were the apparent pKa, the solubility versus pH, the binding affinity with siRNA and the colloidal properties of complexes. Some parameters, like the pKa or the binding enthalpy with siRNA, showed a marked transition from DP 5 to DP 13, suggesting that electrostatic properties of COS vary considerably in this range of DP. The colloidal properties of siRNA/COS complexes were affected in a different way by the COS chain length. In particular, COS of relatively high DP (≥50) were required to form small complex particles with good stability.