Folding and assembly of artificially designed superhelical proteins

• Applicant: Professor Dr. Johannes Stigler
• Subject Area: Biophysics
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 527129816

Proteins based on tandem repeats of small sequence motifs are ubituitous in nature but have also important applications in bionanotechnology. Recent advances in artificial intelligence have driven enormous successes in the de-novo design of protein structures, many of which are built upon small tandem repeats that fold into solenoidal shapes, like their natural counterparts. These solenoidal building blocks can be designed to further self-assemble into larger complexes, for the construction of nanoscale machines, or to serve as scaffolds for the design of ligand-binders in the context of nanomedicine. While the folding and assembly of proteins has been studied for decades, there is a lack of experimental data on whether our current picture of how repeat proteins fold also pertains to the realm of these novel de-novo designed proteins. For example, because most design pipelines only optimize for the thermomodynamic stability of the folded structure, but not for an efficient pathway of folding, it is possible that the artificial design process causes frustrations in the protein folding energy landscape that may result in inefficient (i.e., slow) folding, as well as mechanically rigid structures. Furthermore, while the mechanics of some naturally occuring repeat proteins have been studied, there is little information on the mechanics of de-novo designed repeat proteins. To close this gap in knowledge, I propose studying the folding and assembly of artificially designed solenoid repeat proteins with mechanical unfolding experiments by optical tweezers at the single molecule level. We have previously demonstrated that similar experiments were successful to observe frustrated folding in a de-novo designed non-tandem-repeat protein, and determine the folding pathway of long repeat proteins, at the resolution of single helices. The planned experiments will extend our studies to de-novo designed repeat proteins. They will allow us to follow the folding and assembly of these structures at a maximum resolution of quarter-helices at 10 kHz bandwidth, and simultaneously provide information on the mechanics of the folded structures. The work program will investigate if repeat proteins obtained from artificial design fold and assemble differently from naturally evolved solenoid repeat proteins. In addition, the proposed research will study if modifications of sequence and shape lead to altered flexibility of the folded protein scaffold. These data will be relevant for the future design of dynamic protein structures in biomedicine and nanotechnology.

• DFG Programme: Research Grants