SEARCHING FOR UNIDENTIFIED EARLY INTERMEDIATES IN THE E46Q PYP PHOTOCYCLE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photoactive yellow protein (PYP) from Halorhodospira halophila is a blue-light photoreceptor whose chromophore is a 4-hydroxycinnamic acid which is covalently linked by a thioester bond to the ¿-sulfur of Cys69. Due to its small size (14kDa) and the ease of producing well-diffracting crystals, PYP has served as an excellent model system for studying signal transduction in molecular detail. Previous Laue diffraction studies (1-4) on single PYP crystals with nanosecond (ns) and longer time resolution have identified structurally distinct intermediate species which can be correlated with those observed spectroscopically. Here we aim to study the structural changes of early intermediates in the photocycle of the mutant E46Q PYP. This mutant is one of most interesting mutants of PYP, because it has no hydrogen bonds between the chromophore and the nearby Glu46 residue, thus it is known to have a faster photocycle than the wild type PYP (5-7). The potential cryo-trapped intermediates (8) and the Laue crystallographic structural dynamics from 10 ns to 100 ms (9, 10) of E46Q provides the insight of the sub-10 ns intermediates to be less stable than the early ps intermediates found in wt PYP because of the weaker hydrogen bond. These early intermediates of E46Q might be also expected to have shorter lifetimes than that of wt-PYP, perhaps in the hundreds of picosecond (ps) range. This hypothesis is supported by the refinement of cryotrapped intermediates, which show the early intermediate and pRCW-like species in both wild-type and E46Q PYP (3, 8). Thus we propose to build on our successes in the past, improve the quality of the experimental results, and consolidate our results. New data with improved signal to noise ratio from more efficient laser excitation with stretched laser pulses should allow us to characterize, for the first time, the structure of the primary intermediate and it structural dynamics generated during the E46Q PYP photocycle.