Biology subjects

Bryan, P. N.

Publications and source records attributed to Bryan, P. N..

2 recordsLinked to original sources

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↗

bioRxiv2020

Engineering protein-specific proteases: targeting active RAS

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

biochemistry↗