5YXI

Designed protein dRafX6

Summary for 5YXI

• Entry DOI: 10.2210/pdb5yxi/pdb
• NMR Information: BMRB: 36136
• Descriptor: Designed protein dRafX6 (1 entity in total)
• Functional Keywords: designed protein, de novo protein
• Biological source: synthetic construct
• Total number of polymer chains: 1
• Total formula weight: 8763.37

Authors

Liu, R. (deposition date: 2017-12-05, release date: 2018-12-05, Last modification date: 2024-05-15)

Primary citation

Liu, R.,Wang, J.,Xiong, P.,Chen, Q.,Liu, H.
De novo sequence redesign of a functional Ras-binding domain globally inverted the surface charge distribution and led to extreme thermostability.
Biotechnol.Bioeng., 118:2031-2042, 2021

PubMed Abstract: To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras-binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras-binding interface. Directed evolution of the redesigned RBD improved its Ras-binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non-interfacial surface residues at once, ABACUS redesign was able to globally "invert" the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems.

PubMed: 33590881
DOI: 10.1002/bit.27716

Experimental method

SOLUTION NMR