Site-Specific Mutations Provide New Insights into the Origin of pH Effects and Alternative Spectral Forms in the Photoactive Yellow Protein from Halorhodospira halophila

Abstract

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.

pH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore

“…Though generally pB dark is considered the low pH form of pG, the high pH form of pG is a pB dark form as well. At very low and very high pH values one also has to take into account chromophore hydrolysis (34,37).…”

Five-dimensional crystallography

“…This was achieved by changing the temperature by only 10 K. The temperature range in which these experiments will be feasible spans from the freezing point of the liquor in the crystals which should be substantially below 273 K to the temperature where crystal stability is compromised by protein denaturation. PYP melts in solution at around 358 K (Meyer et al, 2003). In crystals the melting temperature could be even larger.…”

Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface

“…These interactions have been studied by both site-directed mutagenesis (18-21) and computational approaches (24-27) (SI Text). These studies have revealed Tyr42 and Glu46 as the two dominant side chains in the spectral tuning of PYP (18)(19)(20)(21)(24)(25)(26)(27)(28). Weakening of the Glu46-pCA hydrogen bond (29,30) in the E46Q mutant results in a red-shift to 460 nm, while complete disruption of the hydrogen bond in the E46V mutant results in a further red-shift to 478 nm (21).…”