Functional studies on the n-terminal region of photoactive yellow protein

Abstract

Stimulus-induced release of helical elements attached to a PAS core is a recurring feature in PAS domain protein signaling. Photoactive yellow protein (PYP) is a bacterial PAS domain photoreceptor, which contains p-coumaric acid (pCA) as its chromophore, and serves as a model system for such conformational dynamics. Deletion of the N-terminal 25-residue helical extension in PYP slows down the decay of the pB signaling state in the PYP photocycle ~1850-fold. Here we explore the mechanism by which the N-terminal region facilitates pB decay in the protein. The addition of a synthetic peptide corresponding to the N-terminal 25 residues to the delta25 PYP mutant accelerates pB decay, but only by a factor ~4 and with poor peptide affinity (~10 mM). The strong pH-dependence of this peptide effect implies a key role for electrostatic interactions between the negatively charged N-terminal region and the positively charged PAS core, a conserved feature in the PYP family. Our results imply a critical role for the covalent tethering of the N-terminal region by increasing local concentration and by causing directional collisions that avoid non-productive associations. Unexpectedly, attachment of an N-terminal His-tag to delta 25 PYP accelerates pB decay, implying low sequence specificity for the functioning of the N-terminal region. Progressive deletion of the N-terminal extension indicates that its secondary structure only modestly affects pB decay. These results imply that the strong variation in pB decay kinetics observed in the PYP family results mainly from substitutions in the PAS core, and that non-specific but directional and electrostatically guided molecular collisions between the N-terminal extension and the PAS core control the signaling kinetics in PYP.