Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida:: a paradigm for an extended LOV construct

The open reading frame PP2739 from Pseudomonas putida KT2440 encodes a 151 amino acid protein with sequence similarity to the LOV domains of the blue- light sensitive protein YtvA from Bacillus subtilis and to the phototropins ( phot) from plants. This sensory box LOV protein, PpSB2- LOV, comprises a LOV core, followed by a C- terminal segment predicted to form an alpha- helix, thus constituting a naturally occurring paradigm for an extended LOV construct. The recombinant PpSB2- LOV shows a photochemistry very similar to that of YtvA and phot- LOV domains, yet the lifetime for the recovery dark reaction, tau(rec) = 114 s at 20 degrees C, resembles that of phot- LOV domains ( 5 - 300 s) and is much faster than that of YtvA or YtvA- LOV ( 43000 s). Time- resolved optoacoustics reveals phot- like, light- driven reactions on the ns - mu s time window with the sub- nanosecond formation of a. flavin triplet state ( Phi T = 0.46) that decays into the. flavin - cysteine photoadduct with 2 ms lifetime ( Phi 390 = 0.42). The. fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full- length YtvA, although the quantum yield, Phi F, is smaller ( about 55% of YtvA) due to an enhanced static quenching efficiency. The anisotropy of W97 is the same as for W103 in YtvA ( 0.1), and considerably larger than the value of 0.06, found for W103 in YtvA- LOV. Different to YtvA and YtvA- LOV, the. fluorescence for W97 becomes larger upon photoproduct formation. These data indicate that W97 is located in a similar environment as W103 in full- length YtvA, but undergoes larger light- driven changes. It is concluded that the protein segment located C- terminally to the LOV core ( analogous to an interdomain linker) is enough to confer to the conserved tryptophan the. fluorescence characteristics typical of full- length YtvA. The larger changes experienced by W97 upon light activation may reflect a larger conformational freedom of this protein segment in the absence of a second domain.