pH-dependent Equilibrium between Long Lived Near-UV Intermediates of Photoactive Yellow Protein

Abstract

Photoactive yellow protein (PYP) 3 (1) is a water-soluble photoreceptor protein suggested to be involved in the negative phototaxis of Halorhodospira halophila (2). PYP is an attractive model protein for a common signaling mechanism because it is a structural prototype of the PAS domain superfamily, most of which are involved in the biological sensing (3). PYP is composed of 125 amino acid residues and the p-coumaric acid chromophore linking to the cysteine residue by a thioester bond (4 -6). In the dark state, the phenolic oxygen of the chromophore is deprotonated (5) and hydrogen bonded with protonated Tyr-42 and Glu-46 (see Fig. 1), resulting in the yellow color. Photon absorption by the chromophore initiates the photocycle of PYP (7-11). The primary photochemical event of PYP is the trans-cis isomerization of the chromophore (12, 13), like bacterial retinal proteins. Several intermediates are then formed, followed by a return to the dark state in 100 ms under physiological conditions. During the photocycle, photon energy stored in the twisted chromophore is released to the protein moiety to form the putative signaling state (called PYP M , I 2 , or pB). Because of a largely blue-shifted absorption spectrum with respect to the dark state, as well as the long lifetime (ϳ100 ms), PYP M is easily detectable by UV-visible spectroscopy. This spectral blue shift is considered to be caused by protonation of the phenolic oxygen of the chromophore (5,14).Infrared spectroscopy demonstrated that PYP M is formed from its precursor (PYP L , I 1 , or pR) in two steps (15, 16). First, a proton at Glu-46 is transferred to the chromophore, but the global conformational change of the protein moiety does not take place at this stage, as shown by a small absorbance change of the amide mode. This intermediate (pBЈ) has a protonated chromophore, but the chromophore binding site is considered to be preserved. Second, a global conformational change takes place to form PYP M . As the lifetime of pBЈ (ϳ1 ms) is not markedly different from pR (ϳ100 s) and the absorption spectra of PYP M and pBЈ are similar, isolation of pBЈ has been difficult in steady-state measurement. Recently, the presence of pBЈ between PYP L and PYP M has been established by time-resolved UV-visible spectroscopy and Raman spectroscopy (17)(18)(19).Although the signaling pathway of the PYP system has not yet been identified, detailed characterization of long lived intermediates (signaling state) is essential for understanding the signal transduction mechanism of PYP. We have demonstrated extensive conformational change upon formation of PYP M . The dimension of PYP M is significantly larger than the dark state as shown by ϳ1 Å increase of R g (20,21) and PYP M is in a partially unfolded state (22). The N-terminal cap and the central part undergo a substantial structural change (21, 23); however, the cause of this large conformational change has not yet been determined.Accumulated evidence suggests that properties of PYP M are highly pH-sensitive. The extent of con...

pH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore

“…5). Basically, these results confirm previous work (18,25). At low pH pB has an absorption maximum of 368 nm, at medium pH an absorption maximum of 355 nm, and at high pH an absorption maximum of 430 nm, with corresponding pK a values at 5.9 and 9.9, respectively.…”

“…Furthermore, the pB intermediate has been shown to have absorption characteristics that depend on conditions such as pH, to the extent that it can be highly similar to pBЈ. Based on available infrared difference spectra of both pBЈ (17) and a pB intermediate with UV/visible absorption characteristics similar to pBЈ (18), we conclude that pBЈ and this pB are distinct intermediates. In this study, we will assume a unidirectional reaction between pBЈ and pB.…”

“…Another important characteristic of PYP is that, upon formation of the putative signaling state pB, the protein undergoes a large structural change (17,24). This characteristic, however, is also pH-dependent, such that at lower pH the extent of structural change decreases (18). The two different structural forms of pB have different absorption spectra.…”

Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation

“…25,26 A wide range of techniques have demonstrated that the formation of the signalling state of PYP is accompanied by large structural changes that lead to partial unfolding of the protein. 20,23,24,[27][28][29][30][31][32][33] These large structural changes are initiated by intra-molecular proton transfer and presumably assist in altering interactions with a downstream transducer.…”

“…The signalling state of PYP exists in multiple subspecies of which the degree of population depends on pH. 31 At pH 8 a 'medium pH' pB sub-species is dominant, while at pH 6 also a significant amount of the 'low-pH' pB species ( pK 5.9) is present. 52 The 'low pH' pB sub-species presumably is unfolded to a smaller extent than the 'medium pH' pB sub-species.…”