Global Conformational Changes upon Receptor Stimulation in Photoactive Yellow Protein

Abstract

Biological signal transduction starts with the activation of a receptor protein. Two central questions in signaling are the mechanism of activation by a stimulus and the nature and extent of the protein conformational changes involved. We report extensive evidence for the occurrence of large structural changes upon the light activation of photoactive yellow protein (PYP), a eubacterial photosensor. Absorption of a blue photon by the p-coumaric acid (pCA) chromophore in pG, the initial state of PYP, results in the formation of pB, a putative signaling state. In the presence of an adequate hydration shell, large structural changes in the protein backbone, involving both solvent accessible and core regions, were detected using Fourier transform infrared (FTIR) difference spectroscopy. A significant part (23%) of the amide groups which are buried in pG become exposed to the solvent in pB, as measured through light-induced H/D exchange, using both electrospray ionization mass spectrometry and FTIR spectroscopy. Exposure of previously buried hydrophobic sites would lead to an increase in heat capacity during pB formation and a decrease in heat capacity during pB decay. Thermodynamic studies indeed show that the heat capacity change of pB activation is -2.35 ( 0.08 kJ/(mol/K), independent of pH from pH 2.4-7.5. A model for photoactivation of PYP is proposed, which provides a framework for a deeper understanding of receptor activation in general.Biological signal transduction is of paramount importance for the functioning of living organisms. The first component in signal transduction chains is a receptor protein. Stimuli from either within the cell or from its surroundings can convert the receptor into its active conformation: the signaling state. The change in receptor protein conformation that occurs during signaling state formation is thought to be responsible for relay of the signal to the next component in the signal transduction chain, involving protein-protein interactions. To obtain a full understanding of the molecular mechanism of signal transduction, it is essential to study the extent and nature of the structural changes that occur during the formation of the signaling state.In most cases formation of the signaling state is caused by the binding of a ligand. Activation of photoreceptors is triggered by the absorption of a photon. The light-activated nature of photoreceptors and the often-observed reversibility of the changes elicited are strong experimental advantages, allowing time-resolved studies and the application of various forms of spectroscopy. Therefore, the most extensive information on receptor activation has been obtained on two photosensory proteins: mammalian rod rhodopsin (1) and sensory rhodopsin I from the Archaeon Halobacterium salinarum (2). However, detailed structural studies on the conformational changes that occur during receptor activation have not been reported and are hampered by the lack of highresolution structural data on these proteins. We employ photoactive yellow pr...

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

“…3). This dispersed distribution is in line with the partially unfolded nature of the pB state (25)(26)(27)(28)(29): Its decay involves refolding of many regions of the protein, and can thus be affected by mutations at many different locations. The statistically significant correlation of 0.59 between ΔG U and τ pB in the Ala scan dataset (Table S4) supports a link (25) between protein folding and pB decay.…”

“…Photoexcitation causes the conversion of the initial pG dark state of PYP into the long-lived pB state. The pB state has a blueshifted absorbance spectrum and is partially unfolded (25)(26)(27)(28)(29). Its formation involves dissociation of the N-terminal region from the core of PYP (30,31).…”

Axis-dependent anisotropy in protein unfolding from integrated nonequilibrium single-molecule experiments, analysis, and simulation

“…The structure of PYP without the first 25 residues (termed ⌬25PYP) in the pB state has recently been investigated by NMR experiments (41) and parallel tempering molecular dynamics (MD) simulations (42). This structure is consistent with spectroscopic studies [FTIR (32), NMR (35,43,44), H/D exchange (37,45), fluorescence (46), and circular dichroism (40)] of the pB state in aqueous solution. That is, PYP partially unfolds its PAS domain during the photocycle, and this may be a key process in its signaling function (47).…”

Protonation/Deprotonation Reactions Triggered by Photoactivation of Photoactive Yellow Protein from Ectothiorhodospira halophila

“…1A). Because it has been demonstrated that PYP exposes hydrophobic contact surface when it is present in the pB state (28,29) and pH indicators might bind to such sites, similar measurements were performed in a solution buffered with 50 mM MES buffer (pH 6.0). Fig.…”