Kauffman, T. | bioRxiv Science

bioRxiv2022

Design and characterization of a protein fold switching network

Protein sequences encoding three common small folds (3, {beta}-grasp, and /{beta}-plait) were connected in a network with high-identity intersections, termed nodes. The structures of proteins around nodes were determined using NMR spectroscopy and analyzed for stability and binding function. To generate nodes, the amino acid sequence encoding a shorter fold (3a or {beta}-grasp) is embedded in the structure of the ~50% longer /{beta}-plait fold and a new sequence is designed that satisfies two sets of native interactions. This leads to protein pairs with a 3a or {beta}-grasp fold in the shorter form but an /{beta}-plait fold in the longer form. Further, embedding smaller antagonistic folds in longer folds creates critical states in the longer folds such that single amino acid substitutions can switch both their fold and function. This suggests that abrupt fold switching may be a mechanism of evolving new protein structures and functions.

biochemistry↗

bioRxiv2021

Rules for designing protein fold switches and their implications for the folding code

We have engineered switches between the three most common small folds, 3, 4{beta}+, and /{beta}-plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer ([~]90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus, two-state behavior may result from natures negative design rather than being an inherent property of the folding code. Significance StatementWe establish general rules for designing protein fold switches by engineering dimorphic sequences that link the three most common small folds. The fact that switches can be engineered in arbitrary and common protein folds, sheds light on several important questions: 1) What is the generality of fold switching? 2) What types of folds are amenable to switching? 3) What properties are shared by sequences that can fold into two completely different structures? This work has implications for understanding how amino acid sequence encodes structure, how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. ClassificationBiological Sciences: Biochemistry; Physical Sciences: Biophysics and Computational Biology

biophysics↗