Conformational Changes in a Photosensory LOV Domain Monitored by Time-Resolved NMR Spectroscopy

Harper, Shannon M, Neil, Lori C, Day, Iain J, Hore, P J and Gardner, Kevin H (2004) Conformational Changes in a Photosensory LOV Domain Monitored by Time-Resolved NMR Spectroscopy. Journal of the American Chemical Society, 126 (11). pp. 3390-3391. ISSN 0002-7863

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Phototropins are light-activated kinases from plants that utilize light-oxygen-voltage (LOV) domains as blue light photosensors. Illumination of these domains leads to the formation of a covalent linkage between the protein and an internally bound flavin chromophore, destabilizing the surrounding protein and displacing an a-helix from its surface. Here we use a combination of spectroscopic tools to monitor the kinetic processes that spontaneously occur in the dark as the protein returns to the noncovalent ground state. Using time-resolved two-dimensional (2D) NMR methods, we measured the rate of this process at over 100 independent sites throughout the protein, establishing that regeneration of the dark state occurs cooperatively within a 1.6-fold range of observed rates. These data agree with other spectroscopic measurements of the kinetics of protein/FMN bond cleavage and global conformational changes, consistent with these processes experiencing a common rate-limiting step. Arrhenius analyses of the temperature dependence of these rates suggest that the transition state visited during this regeneration has higher energy than the denatured form of this protein domain despite the fact that there is no global unfolding of the domain during this process.

Item Type: Article
Additional Information: ID helped develop idea for real-time NMR monitoring of the recovery of the protein ground state from the photo-excited (signaling) state, and contributed to the analysis of the results. The technique described provides residue-specific kinetic information on the conformational changes occurring in the protein during photoactivation.
Schools and Departments: School of Life Sciences > Chemistry
Depositing User: Iain Day
Date Deposited: 06 Feb 2012 18:42
Last Modified: 22 Mar 2012 15:37
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