Active-Site pKa Determination for Photoactive Yellow Protein Rationalizes Slow Ground-State Recovery

Abstract

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pK determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow.

Perturbation of Short Hydrogen Bonds in Photoactive Yellow Protein via Noncanonical Amino Acid Incorporation

“…The solution pK a 's of pCA bound to the denatured PYP backbone (9.1) and free glutamic acid (4.25) are significantly different. It is well-known that the proton affinity of a residue can change significantly upon burial in a protein, and NMR titration experiments have shown little or no change in the protonation state of pCA in the folded protein between pH 3.4 and 11.4, 30 implying that the proton affinities of the chromophore and E46 in the folded protein environment are altered from those in solution. QM/MM calculated pK a 's of pCA and E46 in the protein environment, 5.4 and 8.6, respectively, 24 agree with the localized proton placement produced by other QM/MM calculations.…”

Metagenomic analysis of the dynamical conversion of photosynthetic bacterial communities in different crop fields over different growth periods

“…Most of the research on H . halophila focused on the photoactive yellow protein [ 62 , 63 ], but the interactions of these bacteria with plants have not been studied. The role of these strains in plant growth and disease resistance will be interesting.…”

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins

“…37 The titration experiments were performed over a pH range of 9−5 (with intervals of 0.25 to 0.35 pH units) in the absence and presence of 300 mM NaCl, and the resulting chemical shift data were fitted against the Henderson−Hasselbalch equation (see the experimental section in Supporting Information). The chemical shifts of 13 Cγ and 13 Glu 24,39 to follow the chemical shift changes as a function of pH (Figure S3). Figure 3a shows the data and the curve-fitting results, and the individual side chain pK a values that were estimated are shown in Table 1, in which similar trends were observed for MaSp2 and MaSp1.…”