Photoactivatable Proteins

The overarching goal of this work is to determine the molecular mechanisms that couple light absorption to the activation of downstream biological processes in flavoprotein photoreceptors. In collaboration with the laboratory of Pete Tonge at Stony Brook University, we complement static and dynamic X-ray crystallographic experiments with time-resolved spectroscopy to elucidate molecular details with a high level of spatial and temporal resolution. This work will significantly advance our understanding of fundamental signaling events and provide a foundation for optogenetic tools. We are also using our novel approach for time-resolved serial crystallography (CSAW), to obtain molecular movies of the dynamic structural features of the photoactive proteins under study. Our current work is focused on the blue light using FAD (BLUF) proteins and the Light Oxygen Voltage (LOV) class of photoreceptors.

appa_paper
The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. (Gil et al. 2016 JACS)

Some of our recent publications in this area:

Agnieszka A. Gil, Sergey P. Laptenok, James N. Iuliano, Andras Kukacs, Anil Verma, Christopher R. Hall, EunBin Yoon, Richard Brust, Gregory M. Greetham, Michael Towrie, Jarrod B. French, Stephen R. Meech, and Peter J. Tonge (2017) Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues. J. Am. Chem. Soc. In press.

Agnieszka A. Gil, Sergey P. Laptenok, Jarrod B. FrenchJames N. Iuliano, Andras Lukacs, Christopher R. Hall, Igor V. Sazanovich, Gregory M. Greetham, Adelbert Bacher, Boris Illarionov, Markus Fischer, Peter J. Tonge and Stephen R. Meech (2017) Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa Lov DomainJ. Phys. Chem. B, 121(5): 1010-1019.

Agnieszka Gil, Allison Haigney, Sergey Laptenok, Richard Brust, Andras Lukacs, James Iuliano, Jessica Meng, Eduard Melief, Rui-Kun Zhao, EunBin Yoon, Ian Clark, Gregory Greetham, Annabelle Ng, James Truglio, Jarrod B. French, Stephen R. Meech, and Peter J. Tonge. (2015) The Mechanism of the AppaBLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: The Effect of the Y21 pKa on the Forward and Reverse Ground State Reactions. J. Am. Chem. Soc., 138: 926-935