Photoactive yellow protein (PYP) is a eubacterial photoreceptor and a structural prototype of the PAS domain superfamily of receptor and regulatory proteins. We investigate the activation mechanism of PYP using time-resolved Fourier transform infrared (FTIR) spectroscopy. Our data provide structural, kinetic, and energetic evidence that the putative signaling state of PYP is formed during a large-amplitude protein quake that is driven by the formation of a new buried charge, COO(-) of the conserved Glu46, in a highly hydrophobic pocket at the active site. A protein quake is a process consisting of global conformational changes that are triggered and driven by a local structural "fault". We show that large, global structural changes take place after Glu46 ionization via intramolecular proton transfer to the anionic p-coumarate chromophore, and are suppressed by the absence of COO(-) formation in the E46Q mutant. Our results demonstrate the significance of buried charge formation in photoreceptor activation. This mechanism may serve as one of the general themes in activation of a range of receptor proteins. In addition, we report the results of time-resolved FTIR spectroscopy of PYP crystals. The direct comparison of time-resolved FTIR spectroscopic data of PYP in aqueous solution and in crystals reveals that the structure of the putative signaling state is not developed in P6(3) crystals. Therefore, when the structural developments during the functional process of a protein are experimentally determined to be very different in crystals and solutions, one must be cautious in drawing conclusions regarding the functional mechanism of proteins based on time-resolved X-ray crystallography.

Photoactive Yellow Protein ( PYP), discovered almost 20 years ago in Ectothiorhodospira (Halorhodospira) halophila, 1 is a 4-hydroxycinnamic acid-containing protein that functions as a blue-light photoreceptor in a behavioral avoidance response in this organism. During the past 10 years, PYP has become a model system for studies in photochemistry and protein folding, to the extent that it has become competitive with the rhodopsins. This is because PYP is small and very water-soluble, forms crystals readily (diffracting to high resolution), and shows excellent chemical-and photo-stability. These overall characteristics have allowed the application of an array of physicochemical techniques to analyze the biological function of PYP, i.e., the translation of a change of the configuration of its 4-hydroxycinnamic acid chromophore into an altered conformation of the surrounding protein. This has led to detailed insight into this process, both temporally and spatially, with respect to the structure of the transient intermediates involved, although we are still quite far from being able to track the position of all atoms in space, upon light activation of the protein in the relevant time domain. Nevertheless, the data already obtained may function as a calibration set in future work, to extend the time span of molecular dynamics simulations of conformational transitions in proteins to the time scale relevant for catalytic turnover. Occasionally, the application of multiple biophysical techniques has led to (seemingly) conflicting results. In one example, this has revealed the fact that the light-induced conformational transitions in this photoreceptor protein can become restricted by the mesoscopic context, e.g., via a crystal lattice. Other inconsistencies, such as those regarding the radius of gyration of the protein, still remain to be explained. Below, we discuss the spatial and temporal details of the series of steps initiated in PYP by a short pulse of blue light, as revealed with this array of biophysical techniques, thereby highlighting contributions from our own group.

In this study we have investigated binding of the fluorescent hydrophobicity probe Nile Red to the photoactive yellow protein (PYP), to characterize the exposure and accessibility of hydrophobic surface upon formation of the signaling state of this photoreceptor protein. Binding of Nile Red, reflected by a large blue shift and increase in fluorescence quantum yield of the Nile Red emission, is observed exclusively when PYP resides in its signaling state. N-terminal truncation of the protein allows assignment of the region surrounding the chromophore as the site where Nile Red binds to PYP. We also observed a pH dependence of the affinity of Nile Red for pB, which we propose is caused by pH dependent differences of the structure of the signaling state. From a comparative analysis of the kinetics of Nile Red binding and transient absorption changes in the visible region we can conclude that protonation of the chromophore precedes the exposure of a hydrophobic surface near the chromophore binding site, upon formation of the signaling state. Furthermore, the data presented here favor the view that the signaling state is structurally heterogeneous.