A Hinge Migration Mechanism Unlocks the Evolution of Green-To-Red Photoconversion in GFP-Like Proteins

Abstract

In proteins, functional divergence involves mutations that modify structure and dynamics. Here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral GFP-like proteins, and their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The photoconversion-competent (red) phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. The mutational sites appear allosterically coupled to the dampening region, while providing conformational mobility to active site residues via epistasis.We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to provide red color. Photoconversion rate measurements provide a bell-shaped curve, indicating that the reaction is controlled by the two apparent pKa values 4.5 (± 0.2) and 7.5 (± 0.2) flanking the chromophore pKa of 6.3 (± 0.1). We tentatively assign these values to the salt-bridged residues Glu222(211) and His203(193), and suggest that reverse protonation may enhance light-induced active site remodeling. In combination, the crystallographic, dynamic and kinetic data support a mechanism that utilizes light to coordinate the transient enhancement of Glu222 proton affinity and His65(63) alpha-carbon acidity, suggesting a concerted process of proton abstraction and main-chain bond scission. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities. (This work was supported by NSF Grant No. MCB-0615938 to R. M. W. and NIH Grant No. U54 GM094599 to R. M. W. and S. B. O.).