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Recording of Blue Light-Induced Energy and Volume Changes within the Wild-Type and Mutated Phot-LOV1 Domain from Chlamydomonas reinhardtii

Aba Losi ^* , Tilman Kottke  and Peter Hegemann 

^* Department of Physics, University of Parma-Istituto Nazionale per la Fisica della Materia, 43100, Parma, Italy; Max-Planck-Institut für Bioanorganische Chemi, 45470 Mülheim an der Ruhr, Germany; Institut für Physikalische und Theoretische Chemie, Universität Regensburg, 93040 Regensburg, Germany; and Institut für Biochemie, Universität Regensburg, 93040 Regensburg, Germany

Correspondence: Address reprint requests to Aba Losi, Dept. of Physics, University of Parma-Istituto Nazionale per la Fisica della Materia, Parco Area delle Scienze 7/A, 43100, Parma, Italy. E-mail: losia{at}fis.unipr.it.

The time-resolved thermodynamics of the flavin mononucleotide (FMN)-binding LOV1 domain of Chlamydomonas reinhardtii phot (phototropin homolog) was studied by means of laser-induced optoacoustic spectroscopy. In the wild-type protein the early red-shifted intermediate LOV₇₁₅ exhibits a small volume contraction, V₇₁₅ = -1.50 ml/mol, with respect to the parent state. LOV₇₁₅ decays within few µs into the covalent FMN-Cys-57 adduct LOV₃₉₀, that shows a larger contraction, V₃₉₀ = -8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV₃₉₀, E₃₉₀ = 180 kJ/mol, ensures the driving force for the completion of the photocycle and points to a strained photoreceptor conformation. In the LOV-C57S mutated protein the photoadduct is not formed and V₃₉₀ is undetected. Large effects on the measured Vs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with V₃₉₀ = -2.0 and -1.9 ml/mol, respectively, and V₇₁₅ 0. The D31Q and D31N substitutions exhibit smaller but well-detectable effects. These results show that the photo-induced volume changes involve the protein region comprising Arg-58, which tightly interacts with the FMN phosphate group.

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