The Two Photocycles of Photoactive Yellow Protein fromRhodobacter sphaeroides

Abstract

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

Identification of Six New Photoactive Yellow Proteins—Diversity and Structure–Function Relationships in a Bacterial Blue Light Photoreceptor†

“…Finally, the lifetime of the pB state varies by more than 6 orders of magnitude—from 1 ms in Rb. sphaeroides (41) to longer than an hour in S. ruber . This raises the question as to how different members of the PYP family tune the p CA absorbance maximum, p CA p K a and pB lifetime over a wide array of values to achieve biologically functional values.…”

“…p CA in the PYP from T. tepidum exhibits an even higher p K a —10.2 (39). Also, the lifetime of the pB state varies significantly among the PYPs: from 1 ms for the PYPs from the two Rhodobacter species (10,40,41) to minutes for the PYPs from R. centenum and T. tepidum and the second PYP from H. halophila (9,12,39).…”

Photokinetic, Biochemical and Structural Features of Chimeric Photoactive Yellow Protein Constructs

“…Haker et al . created a model of Rb. sphaeroides PYP in which they also noted increased exposure of the chromophore due to T50A and M100G substitutions.…”

“…capsulatus and Rb. sphaeroides PYP than in the prototypical Hhal PYP (13,16). Interestingly, both Rcaps and Rsph PYP have an M100G substitution in their sequences, which would seem to demand an order of magnitude slower recovery.…”

Photoactive Yellow Protein, the Xanthopsin

“…For PYP from Rb. sphaeroides it has been shown that, upon excitation, the species at 360 can also enter a photocycle, independent from the photocycle of the 446 nm species (Haker et al 2003). Furthermore, in both proteins, the ground state is recovered after photoexcitation ∼100-fold faster than in PYP from H. halophila.…”