Structural Insight into Split Green Fluorescent Protein

Abstract

Split green fluorescent proteins (GFP) with a structural element removed have potentially useful applications in the control of light-driven protein-protein interactions, and in the preparation of semi-synthetic proteins with novel spectroscopic or functional properties. The truncated GFP generated by removing the 11th β -strand has been previously shown to reassemble with synthetic strand 11 peptide only following light activation (Kent and Boxer, JACS 2011). The light irradiation is thought to drive a photo-isomerization reaction within the chromophore of the protein, switching the truncated protein from the thermally stable trans-like state to the strand-receptive cis-like state. However, neither the process by which this occurs nor the presumed conformational changes that accompany it are understood to any level of structural detail. We utilize the unique reassembly behavior of split GFP in conjunction with solution state NMR to gain some insight into the structure and dynamics of truncated GFP. The rates of hydrogen-deuterium exchange (HDX) of the amide protons in the truncated GFP compared to the rates in an intact GFP (Huang, et al. and Jackson, JMB 2007) serve as a proxy for solvent exposure and protein stability. The HDX experiments show evidence of residual structure in the truncated GFP, based on assignments that correspond to peaks in intact GFP. Obtaining further structural information will be instrumental in solidifying our understanding of the observed phenomena, and perhaps lead to the rational design and engineering of other split fluorescent protein systems with interesting photo-reactive behaviors.