The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N-or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by Ϸ3 nm (at the 10-pN detection limit of the measurement) and destabilized by Ϸ30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulusinduced partial protein unfolding can be used as a signaling mechanism.atomic force spectroscopy ͉ photoactive yellow protein ͉ receptor activation B iological signaling starts with the activation of receptor proteins, in which stimuli trigger protein conformational changes that relay a signal to an associated signal transduction chain. A central question concerns the nature of the structural changes that activate a receptor protein. The mechanism of signaling by PerArnt-Sim (PAS) domain-containing proteins is of considerable interest, because these structural domains occur in many signaling proteins (1). In addition, incorrect signaling by PAS domains is associated with a range of diseases. Important examples are mutations in the human ERG potassium channel, which causes cardiac arrhythmias (2), and the involvement of hypoxia-inducible factors in myocardial and cerebral ischemia (3).We investigate the photoinduced structural changes in the bacterial blue light receptor, photoactive yellow protein (PYP), a prototype for PAS domains (4). As shown in Fig. 1, PYP consists of two N-terminal ␣-helices (residues 1-25), followed by a typical PAS domain (1, 5) fold (residues 26-125) that contains a central six-stranded ␤-sheet flanked by ␣-helices (6). The protein exhibits photochemistry based on its p-coumaric acid chromophore (7). Blue light converts the initial pG state of PYP into the photoactivated pB state in milliseconds, whereas pB decays back to pG in Ϸ0.5 s (8). The pB state is believed to trigger negative phototaxis in purple bacteria (9).The structural changes that occur upon pB formation involve partial unfolding of the receptor, indicating partial protein unfolding as a novel signaling m...

Halorhodospira halophila is among the most halophilic organisms known. It is an obligately photosynthetic and anaerobic purple sulfur bacterium that exhibits autotrophic growth up to saturated NaCl concentrations. The type strain H. halophila SL1 was isolated from a hypersaline lake in Oregon. Here we report the determination of its entire genome in a single contig. This is the first genome of a phototrophic extreme halophile. The genome consists of 2,678,452 bp, encoding 2,493 predicted genes as determined by automated genome annotation. Of the 2,407 predicted proteins, 1,905 were assigned to a putative function. Future detailed analysis of this genome promises to yield insights into the halophilic adaptations of this organism, its ability for photoautotrophic growth under extreme conditions, and its characteristic sulfur metabolism.