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

Abstract

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.

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein

“…What is clear is that LOV domains all share similar architectures and photochemical responses to illumination, harnessing the energy of incoming blue light photons to form a covalent adduct between the Sγ sulfur on a conserved cysteine residue and the C4a carbon of a flavin cofactor (12,13). Formation of this bond generates structural changes that propagate to the domain surface, altering the interactions of the core LOV domain with intra-or interprotein partners (14)(15)(16)(17)(18). For example, structural studies on Avena sativa phototropin 1 LOV2 (AsLOV2) demonstrated light-induced unfolding of the Jα-helix located C-terminal to the canonical LOV domain (15).…”

Photosensitivity of Kinase Activation by Blue Light Involves the Lifetime of a Cysteinyl-Flavin Adduct Intermediate, S390, in the Photoreaction Cycle of the LOV2 Domain in Phototropin, a Plant Blue Light Receptor

“…The half-lives of the native phot and the R58K substitute were 204 and 73 s, respectively (32). To alter the lifetime of S390 in P1L2K, Arg-513 (supplemental Fig.…”

Conformational Switching in the Fungal Light Sensor Vivid

“…Both small and large amplitude conformational changes have been implicated in signal propagation by PAS domain proteins (2,21,28,(30)(31)(32); however, it has been challenging to link conformational changes to a biological function in vivo. We performed in vivo complementation studies of VVD Cys 71 Ser in Neurospora to demonstrate that light-induced conformational changes involving the N-terminal cap are essential for cellular function.…”