Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations

Abstract

Classical Anfinsen′s dogma states that a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, recent advances have revealed that single amino acid sequences can fold into two or more conformations. Here, we propose a novel approach to design a protein that adopts interconvertible alternative functional conformations, termed ″seesaw″ protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal β-strand and DHFR lacking the N-terminal β-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analysis, including AFM imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. In addition, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a new direction for creating artificial proteins with on-off behaviors.