Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae.

Background: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state.

Structural basis for the slow dark recovery of a full-length LOV protein from Pseudomonas putida.

Blue-light photoreceptors containing light–oxygen–voltage (LOV) domains regulate a myriad of different physiological responses in both eukaryotes and prokaryotes. Their light sensitivity is intricately linked to the photochemistry of the non-covalently bound flavin mononucleotide (FMN) chromophore that forms a covalent adduct with a conserved cysteine residue in the LOV domain upon illumination with blue light. All LOV domains undergo the same primary photochemistry leading to adduct formation; however, considerable variation is found in the lifetime of the adduct state that varies from seconds to several hours.

Flavin mononucleotide-based fluorescent reporter proteins outperform green fluorescent protein-like proteins as quantitative in vivo real-time reporters.

Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

Mutual exchange of kinetic properties by extended mutagenesis in two short LOV domain proteins from Pseudomonas putida.

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.

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus.

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins.

Reporter proteins for in vivo fluorescence without oxygen.

Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage-sensing domains, we engineered flavin mononucleotide-based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.