A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We used protein engineering and atomic force microscopy to explore the single-molecule mechanics of the figure-eight knot in the chromophore-binding domain of the red/far-red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of approximately 47 pN, whereas phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at approximately 73 pN. These forces are not unusual in mechanical protein unfolding, and thus the presence of the knot does not automatically indicate a superstable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting the sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time (to the best of our knowledge), our experiments allow a direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular-dynamics simulations corroborate this number. Finally, we find that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against a dramatic rearrangement of the native GAF dimer interface upon photoconversion.

Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74-100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes.