Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila.
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.
Meyer
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Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Devanathan
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S
Woo
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Getzoff
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Tollin
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Cusanovich
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D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; C050162:photoactive yellow protein, Bacteria