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

• Published in: Physical chemistry chemical physics : PCCP Vol. 7; no. 14; p. 2804
• Main Authors: Krauss, U, Losi, A, Gärtner, W, Jaeger, K-E, Eggert, T
• Format: Journal Article
• Language: English
• Published: England 01-01-2005
• Subjects: Bacillus subtilis - metabolism; Bacillus subtilis - radiation effects; Cryptochromes; Flavoproteins - chemistry; Light; Oxygen - chemistry; Photochemistry; Protein Structure, Secondary; Pseudomonas putida - chemistry; Pseudomonas putida - radiation effects; Spectrometry, Fluorescence; Tryptophan - chemistry
• ISSN: 1463-9076
• DOI: 10.1039/b504554a

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, taurec=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 (>3000 s). Time-resolved optoacoustics reveals phot-like, light-driven reactions on the ns-micros time window with the sub-nanosecond formation of a flavin triplet state (PhiT=0.46) that decays into the flavin-cysteine photoadduct with 2 micros lifetime (Phi390=0.42). The fluorescence spectrum and lifetime of the conserved W97 resembles the corresponding W103 in full-length YtvA, although the quantum yield, PhiF, 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.