Recording of blue light-induced energy and volume changes within the wild-type and mutated phot-LOV1 domain from Chlamydomonas reinhardtii.
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 LOV(715) exhibits a small volume contraction, DeltaV(715) = -1.50 ml/mol, with respect to the parent state. LOV(715) decays within few micro s into the covalent FMN-Cys-57 adduct LOV(390), that shows a larger contraction, DeltaV(390) = -8.8 ml/mol, suggesting a loss of entropy and conformational flexibility. The high energy content of LOV(390), E(390) = 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 DeltaV(390) is undetected. Large effects on the measured DeltaVs are observed in the photochemically competent R58K and R58K/D31Q mutated proteins, with DeltaV(390) = -2.0 and -1.9 ml/mol, respectively, and DeltaV(715) approximately 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.
Losi
Aba
A
Department of Physics, University of Parma-Istituto Nazionale per la Fisica della Materia, 43100, Parma, Italy. losia@fis.unipr.it
Kottke
Tilman
T
Hegemann
Peter
P
D019943:Amino Acid Substitution; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D056931:Cryptochromes; D003624:Darkness; D004307:Dose-Response Relationship, Radiation; D004735:Energy Transfer; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D006207:Half-Life; D008027:Light; D009038:Motion; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation / Q000528:radiation effects; D017434:Protein Structure, Tertiary / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry
D056931:Cryptochromes; D005420:Flavoproteins; D011994:Recombinant Proteins