Erik Sedlák

Recombinant core spidroin eADF4(C16) has received increasing attention due to its ability to form micro‐ and nano‐structured scaffolds, which are based on nanofibrils with great potential for biomedical and biotechnological applications. Phosphate anions have been demonstrated to trigger the eADF4(C16) self‐assembly into cross‐beta fibrils. In the present work, we systematically addressed the effect of nine sodium anions, namely SO 4 2− , HPO 4 2− (Pi), F − , Cl − , Br − , NO 3 − , I − , SCN − , and ClO 4 − from the Hofmeister series on the in vitro self‐assembly kinetics of eADF4(C16). We show that besides the phosphate anions, only kosmotropic anions such as sulfate and fluoride can initiate the eADF4(C16) fibril formation. Global analysis of the self‐assembly kinetics, utilizing the platform AmyloFit, showed the nucleation‐based mechanism with a major role of secondary nucleation, surprisingly independent of the type of the kosmotropic anion. The rate constant of the fibril elongation in mixtures of phosphate anions with other studied anions correlated with their kosmotropic or chaotropic position in the Hofmeister series. Our findings suggest an important role of anion hydration in the eADF4(C16) fibrillization process.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self‐assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro‐ and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils‐based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self‐assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding, and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self‐assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes.

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.