Mutual Exchange of Kinetic Properties by Extended Mutagenesis in Two Short LOV Domain Proteins from Pseudomonas putida

Abstract

We previously characterized a LOV protein PpSB2-LOV, present in the common soil bacterium Pseudomonas putida, that exhibits a plant phototropin LOV-like photochemistry [Krauss, U., Losi, A., Gartner, W., Jaeger, K. E., and Eggert, T. (2005) Phys. Chem. Chem. Phys. 7, 2804-2811]. Now, we have identified a second LOV homologue, PpSB1-LOV, found in the same organism with approximately 66% identical amino acids. Both proteins consist of a conserved LOV core flanked by short N- and C-terminal extensions but lack a fused effector domain. Although both proteins are highly similar in sequence, they display drastically different dark recovery kinetics. At 20 degrees C, PpSB2-LOV reverts with an average time constant of 137 s from the photoequilibrium to the dark state, whereas PpSB1-LOV exhibits an average dark recovery time constant of 1.48 x 10(5) s. Irrespective of the significant differences in their dark recovery behavior, both proteins showed nearly identical kinetics for the photochemically induced adduct formation. In order to elucidate the structural and mechanistic basis of these extremely different dark recovery time constants, we performed a mutational analysis. Six amino acids in a distance of up to 6 A from the flavin chromophore, which differ between the two proteins, were identified and interchanged by site-directed mutagenesis. The amino acid substitution R66I located near the FMN phosphate in LOV domains was identified in PpSB1-LOV to accelerate the dark recovery by 2 orders of magnitude. Vice versa, the corresponding substitution I66R slowed down the dark recovery in PpSB2-LOV by a factor of 10. Interestingly, the interchange of the C-terminal extensions between the two proteins also had a pronounced effect on the dark recovery time constants, thus highlighting a coupling of these protein regions to the chromophore binding pocket.

Fusion of a Flavin-Based Fluorescent Protein to Hydroxynitrile Lyase from Arabidopsis thaliana Improves Enzyme Stability

“…This is in good agreement with findings of previous studies demonstrating that isolated YFP is mainly monomeric with a weak tendency to dimerize at higher concentrations (dissociation constant [K d ] ϭ 0.1 mM) and consequently does not interfere with the formation of the native quaternary structure of the YFP-fused target protein (28). For all FbFPAtHNL fusions, the dimeric organization of the FbFP module (13,14) apparently results in induced higher-order oligomerization, i.e., yielding fusion proteins with variable amounts of an apparently tetrameric species. In comparison, other HNLs, such as those from Prunus species, Linum usitatissimum, and Manihot esculenta (MeHNL), are both active and stable at low pH (29).…”

“…To the best of our knowledge, bacterial FbFPs have so far not been used as fluorescent reporter modules in translational fusions targeting enzymatically active proteins. This issue is of particular interest, since bacterial FbFPs, in contrast to the plant iLOV system, form stable dimers in solution (13,14) which could exert unforeseen adverse effects on folding, activity, and stability of a fused target protein.…”

The photophysics of LOV-based fluorescent proteins — new tools for cell biology

“…To further exploit naturally occurring LOV variants, two novel LOV-based FPs, namely DsFbFP (isolated from the marine α proteobacterium Dinoroseobacter shibae 42 ) and Pp1FbFP (derived from the P. putida LOV protein Pp1SB1-LOV 43,51 ), were created for this study. Both of these two novel FbFPs originate from "short LOV" proteins, a class of LOV receptors that do not possess any fused effector domains.…”

“…Formation of the non-fluorescing LOV photoadduct during the light-triggered photocycle was prevented by substitution of the cysteine that binds to FMN during the photocycle (DsFbFP: Cys72, Pp1FbFP: Cys53) by an alanine as described previously 8 . To avoid confusion with nomenclature, the first described PpFbFP that is based on the PpSB2-LOV-domain 43 , is now designated as Pp2FbFP. Figure 1 depicts the alignment of the amino acid sequences of nine LOV-based FPs under study.…”

“…Plasmids pRhotHi2-DsLOV and pET28a-PpSB1-LOV encompassing the respective LOV photoreceptor genes from Dinoroseobacter shibae (DsLOV) 42 , and Pseudomonas putida (PpSB1-LOV) 43 , were used as templates. The photoactive cysteine residue (C72) of DsLOV was replaced by alanine using overlap extension PCR, performed with the primers DsLOV+NdeI-up:…”