Understanding the structural bases for regulation and signaling in proteins is a central problem in biophysics. What are the active and inactive conformations of a protein and how does a protein traverse a complex energy landscape as it passes from one to another? To answer this, we require the structures of all intermediates and the rates by which they interconvert, i.e. the chemical kinetic mechanism. We also must identify those residues responsible for relaying the structural signal from one part of the protein to the next and how such structural signal is related to a biological signal. In the blue light photoreceptor photoactive yellow protein (PYP) from Halorhodospira halophila, a member of the PAS domain superfamily, absorption of light by its chromophore leads to conformational changes in the protein associated with differential signaling activity as it executes a reversible photocycle associated with red and blue-shifted intermediates. Time-resolved Laue crystallography allows structural snapshots (as short as 150 picoseconds) of high crystallographic resolution (∼1.6 A) to be taken of a protein as it functions. Here we analyze by singular value decomposition comprehensive time resolved crystallographic data sets of wild-type P YP and its E46Q mutant, comprising tens of time points throughout the photocycles spanning nanoseconds to seconds. We identify and refine the structures of distinct intermediates and provide a plausible chemical kinetic mechanism for their interconversion. In both WT and E46Q PYP, a clear structural progression is visible through these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway through conserved residues and results in structural changes near the N-terminus, over 20 A distant from the chromophore. While experiments to test the biological significance of these residues is not possible in PYP from H. halophila, whose signaling partners are yet unidentified, it is possible to test them in the PYP domain from the sensor histidine kinase Ppr from Rhodocista centenaria, whose static crystal structure we have also solved. The combination of these results from time-resolved and conventional static crystallography, along with previous work on PAS domains, are consistent with a general model for PAS domain-mediated signal transduction.
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