The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is present, refolding from the unfolded state proceeds through the putative signaling state of PYP as an on-pathway intermediate. In addition, moderate denaturant concentrations result in the specific unfolding of the signaling state of PYP. Thus, the signaling state is common to the pathways of folding and signaling. This result provides an avenue for the study of protein folding. We demonstrate how this approach can be used to establish whether a folding intermediate is on-pathway or offpathway. The results also reveal transient partial unfolding as a molecular mechanism for signaling.T o become functionally active, a protein needs to fold into its native three-dimensional structure. Protein folding has been investigated extensively, but many of its aspects remain unresolved. The properties of folding intermediates and their role in the folding mechanism as either productive on-pathway intermediates or dispensable off-pathway intermediates have proven to be difficult to unambiguously determine for many proteins (see refs. 1-4). Here evidence is reported for the hypothesis that protein folding and signaling are directly related. We demonstrate that this provides an approach to experimental studies on protein folding, using a small water soluble receptor protein that is activated by an external stimulus: unfolding and refolding reactions can be triggered not only by using traditional rapid mixing methods, but also by activation of the receptor. Thus, two independent pathways are available to populate the same folding intermediate. The existence, properties, and position in the folding pathway of the intermediate as deduced from rapid mixing studies therefore can be directly and independently tested. The use of a photoreceptor in this approach provides the additional benefit that signaling is triggered by exposure of the receptor to light, allowing for time-resolved measurements on the receptor activation process.To study the relationship between protein folding and signaling, we used photoactive yellow protein (PYP) as a model system. PYP is a small water-soluble photoreceptor from purple bacteria (5-7) that displays rhodopsin-like photochemistry (5, 8) based on its unique p-coumaric acid chromophore (9, 10). PYP is a member of the PAS domain family that is involved in regulation, sensing, and the circadian rhythm (11,12). The involvement of PAS domains in a wide range of biological responses has triggered studies on PYP aimed at understanding the signaling mechanism of this ubiquitous signaling module. Absorbance of a photon initiates a photocycle in PYP by the trans to cis photoisomerization of its chromophore (13)(14)(15)(16)...

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