Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein

TO understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which un dergoes a photocycle upon illumination. The active site residues glutamic a cid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond net work with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the main tenance of the thromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ra y structures show that the hydrogen-bonding interactions between the protei n and the chromophore are weakened in the mutants, leading to increased ele ctron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutant s T50V and Y42F, respectively. Both mutants have slightly perturbed photocy cle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics i s similar to wild-type PYP, suggesting that T50 and Y42 are not directly in volved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type ( 29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as show n by UV/ visible and FT Raman spectroscopy. The second chromophore conforma tion gives rise to a shoulder at 391 nm in the UV/visible absorption spectr um and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conform ation. The two conformations in the Y42F mutant can be interconverted by ch aotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence q uantum yield of the mutant Y42F is 1.8% and is increased by an order of mag nitude when compared to the wild type.