Active Site Mutants Implicate Key Residues for Control of Color and Light Cycle Kinetics of Photoactive Yellow Protein

Abstract

To understand how the protein and chromophore components of a light-sensing protein interact to create a light cycle, we performed time-resolved spectroscopy on site-directed mutants of photoactive yellow protein (PYP). Recently determined crystallographic structures of PYP in the ground and colorless I2 states allowed us to design mutants and to study their photosensing properties at the atomic level. We developed a system for rapid mutagenesis and heterologous bacterial expression for PYP apoprotein and generated holoprotein through formation of a covalent thioester linkage with the p-hydroxycinnamic acid chromophore as found in the native protein. Glu46, replaced by Gln, is buried in the active site and hydrogen bonds to the chromophore's phenolate oxygen in the ground state. The Glu46Gln mutation shifted the ground state absorption maximum from 446 to 462 nm, indicating that the color of PYP can be fine-tuned by the alteration of hydrogen bonds. Arg52, which separates the active site from solvent in the ground state, was substituted by Ala. The smaller red shift (to 452 nm) of the Arg52Ala mutant suggests that electrostatic interactions with Arg52 are not important for charge stabilization on the chromophore. Both mutations cause interesting changes in light cycle kinetics. The most dramatic effect is a 700-fold increase in the rate of recovery to the ground state of Glu46Gln PYP in response to a change in pH from pH 5 to 10 (pKa = 8). Prompted by this large effect, we conducted a careful reexamination of pH effects on the wild-type PYP light cycle. The rate of color loss decreased about 3-fold with increasing pH from pH 5 to 10. The rate of recovery to the colored ground state showed a bell-shaped pH dependence, controlled by two pKa values (6.4 and 9.4). The maximum recovery rate at pH 7.9 is about 16 times faster than at pH 5. The effect of pH on Arg52Ala is like that on wild type except for faster loss of color and slower recovery. These kinetic effects of the mutations and the changes with pH demonstrate that both phases in PYP's light cycle are actively controlled by the protein component.

Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction

“…Structural changes in the ␤4-␤5 and ␣3-␣4 loops would be expected to affect photocycle dynamics because of the presence of critical residues in those regions (26,27). As in E-PYP, the ␤4-␤5 loop is flexible in Ppr-PYP, evidenced by high B factors in this region in chains A and B and its disorder in chain C. The disorder of the ␤4-␤5 loop of chain C is caused by a weak packing interaction between it and the N-terminal helices of chain A, which are similarly disordered.…”

“…A R. centenum Ppr knockout showed no defects in phototaxis but had lower levels of expression of chalcone synthase, an enzyme involved in photoprotection through flavonoid biosynthesis (25). Although the PYP domain of Ppr (Ppr-PYP) is 45% identical in sequence to E-PYP and has similar or identical residues to all those suggested to be critical to absorption spectra and photocycle dynamics in E-PYP (26,27), Ppr-PYP has a considerably slower photocycle than E-PYP (25). Furthermore, as Ppr-PYP controls the activity of a histidine kinase, its study affords not only the opportunity to understand the signaling mechanism of PYPs, but of the entire class of PAS-domain-regulated sensor histidine kinases, which are ubiquitous in bacteria (28).…”

On the absorbance changes in the photocycle of the photoactive yellow protein: A quantum-chemical analysis

“…The agreement between theoretical results and experimental data for PYP G seems to point out that the interaction between protein and chromophore, which has been neglected here, is similar in both ground and excited states. Indeed, the effect of mutants on excitation energies has been at most recorded to be 0.1 eV, for instance, when Glu-46 is replaced by Gln (34). On the other hand, in a recent theoretical study of the phenolate anion in the environment of PYP, He et al (42) have shown that the The results are compared to available experimental data.…”

“…Nevertheless, that the amino acid residue Glu-46 acts as the actual proton donor to the chromophore has been seen with IR techniques (32,33). The cycle is completed when the PYP G structure is recovered by means of a protein-mediated thermal process (34).…”

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

“…Recent progress in experimental techniques made it possible to prepare PYP with an isotope-labeled chromophore (9) and a site-directed mutant of PYP (19,27). These techniques have enabled assignment of the vibrational modes.…”