Fig. 5

Biophysical characterization of recombinant (AQ)₁₂NR3 spider silk proteins in the dope phase and as spun fibers. A Light microscopy images of biomimetic spinning dopes (BSDs) (top, I-III) and classic spinning dopes (CSDs) (bottom, IV-VI), both at a concentration of 100 mg/ml. Regions from II, III, V and VI are enlarged to visualize the differences between the systems, where the BSD contains large droplets indicative of LLPS. B DLS of BSD (left) and CSD (right). Samples were taken after 0, 2, 4, and 6 h to follow the assembly process of the spinning solutions. After this, no further changes were observed, and the dope production was completed. During assembly, the concentrations gradually increased. For BSD, the concentrations at 0, 2, 4, and 6 hours were 27, 63, 96, and 177 mg/ml, respectively. For CSD, the concentrations at 0, 2, 4, and 6 hours were 25, 41, 106, and 197 mg/ml, respectively. These results are consistent with light microscopy observations (A), which revealed that dopes prepared with phosphate buffer create droplets significantly larger in size than those created with CSD. C ¹H-¹⁵N HSQC NMR spectrum of fully labeled ¹³C/¹⁵N-(AQ)₁₂NR3 BSD collected at 850 MHz. The bolded amino acids indicate resonance identities with di- and tripeptide repeats on the basis of 3D experiments [37]. Conformation-dependent chemical shifts illustrate that the repetitive core of the silk protein maintains intrinsic disorder in the condensed phase. D ¹H-¹³C CP-MAS SS-NMR spectrum of sparsely labeled ¹³C/¹⁵N-(AQ)₁₂NR3 lyophilized protein (black) and fibers spun from BSD (blue) and CSD (red) dopes. The full spectrum (top) and magnified image of the low ppm region (bottom) are shown. Fibers spun from both dopes have identical β-sheet formation rates of 77% for poly-A domains according to spectral deconvolutions [37]. E 2D ¹H-¹³C HETCOR SSNMR spectrum collected with frequency-switched Lee‒Goldburg (FSLG) homonuclear proton decoupling for CSD (blue) and BSD (red) fibers. Slices were extracted at the tyrosine side chain resonance to illustrate the differences between the ¹H chemical shifts of one of the tyrosine aromatic rings for the two fibers. A downfield ¹H chemical shift in the BSD fibers is observed, illustrating differences in aromatic ring packing for fibers spun from the two dopes. The drift correction offset glitch due to FSLG homonuclear decoupling is indicated with an asterisk (*). Adapted with permission from [37]. Copyright 2023 American Chemical Society