We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned. Reconstitution of the his-tagged purified apo-protein with 4-hydroxy-cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm. This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy. Moreover, reconstitution with the`locked' analogue 7-hydroxy-coumarin-3-carboxylic acid yields only a single absorbance peak at 441 nm. The 446 nm and 360 nm species are part of a temperatureand pH-dependent equilibrium. Photoactivation of the protein leads to formation of a blue-shifted intermediate as in other PYPs, with a 100-fold increased groundstate recovery rate (k pB3pG = 500 s 31 ) compared to E-PYP. ß
Photoactive yellow protein (PYP)1 is a photoreceptor that has been found in several purple bacteria (1). The first, and so far best studied example for this group of blue light receptors, was found in Ectothiorhodospira halophila (E-PYP) (2). The chromophore, responsible for the photophysical properties of PYP, is 4-hydroxy-cinnamic acid that is bound to Cys-69 via a thiolester linkage (3, 4). The crystal structure of this small protein, consisting of 125 amino acids, has been solved to 1.4-Å resolution (5) and shows an ␣/-fold, which has become the prototype for the folding of the Per-Arnt-Sim domain superfamily (6, 7). In the ground state the chromophore is deprotonated and buried in a hydrophobic pocket of the protein where its negative charge is stabilized via a hydrogen bonding network. Absorption of light induces a photocycle in E-PYP, in which isomerization of the chromophore is the initial step, which leads to the formation of several transient intermediates on the femtosecond to nanosecond timescale (8, 9 (14,15).In contrast to the detailed knowledge available for PYP from E. halophila, other photoactive yellow proteins are biophysically poorly investigated. So far, proteins from three other species were purified and basically characterized: (i) PYP from Rhodospirillum salexigens (16), which shares 71% amino acid sequence identity with E-PYP and has virtually the same ground state absorption spectrum ( max ϭ 445 nm) and similar kinetics of photobleaching and recovery (with time constants for pB formation and pG recovery of 85 s and 210 ms, respectively); (ii) PYP-phytochrome-related protein from Rhodospirillum centenum (17), a hybrid-protein, consisting of 884 amino acids, with an N-terminal PYP domain fused to a central phytochrome-like domain and a C-terminal histidine kinase domain. When heterologously expressed and reconstituted with 4-hydroxy-cinnamic acid, Ppr displays an absorbance maximum at 434 nm and is photoactive; bleaching at 434 nm is accompanied by the initial formation of a red-shifted intermediate with a difference absorption maximum at ϳ470 nm, and subsequently a blue-shifted intermediate is formed with a difference absorption maximum at ϳ330 nm. The recovery to the ground state is biphasic with a fast and a very slow component (lifetimes of 0.21 ms and 46 s, respectively); and (iii) PYP from Rhodobacter sphaeroides (R-PYP), which has been characterized in some more detail (18) and is also the subject of this study.Heterologously expressed R-PYP, reconstituted in vitro with 4-hydroxy-cinnamic acid, is a yellow-colored and photoactive protein (18). The main absorption band with a maximum at 446
Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly 47 and Gly 51 as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly 51 may also play a role in PAS domain proteins where it is one of the few conserved residues.The photoactive yellow protein (PYP) 1 is a photoreceptor presumed to be involved in a phototactic response of the bacterium Ectothiorhodospira halophila to intense blue light (1). Its structure reveals an ␣/ fold with the chromophore, pcoumaric acid bound to the protein via a thioester linkage (2) (see Fig. 1). The protein has been shown to undergo a photocycle, linked to isomerization of the chromophore (3-7). The ground state (pG) has a UV/visible absorbance maximum at 446 nm. After absorption of a blue photon, the protein returns from the primary excited state into the first transient groundstate (8 -11), a strongly red-shifted intermediate (12) Recent Laue diffraction and cryo-crystallographic studies on PYP have given insight into the structural events during and after chromophore isomerization (4, 6, 7). Initially, light absorption induces a flipping of the carbonyl group of the chromophore thioester linkage. At room temperature, in concert with this flipping, the vinyl bond of the chromophore isomerizes from trans to cis, leading to an ϳ60°rotation of the aromatic ring of the chromophore (4). At low temperature (Ͻ150 K), the latter process appears to be blocked about halfway during the isomerization process (7). What both Laue diffraction and cryo-crystallography studies have in common, is that the observed structural changes provide a detailed picture of the early structural events in the photocycle in the immediate environment of the chromophore (4, 6, 7). For PYP in solution, formation of the blue-shifted intermediate pB involves a significant conformational transition, which has the characteristics of a (partial) protein unfolding event, as reported by UV/visible and Fourier transform infrared spectroscopy, mass spectrometry, and 1 H-NMR analyses (17-19). These latter observations hint at the presumed signaling function of PYP, which ultimately should result in a negative phototactic response of the bacterium. ...
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