Part:BBa_K4044003
BcLOV4
BcLOV is a light-oxygen-voltage (LOV) flavoprotein. This protein is a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet in mammalian cells [1]. This protein contains flavin as a chromophore bound to sensory domain Per-Arnt-Sim type (PAS). The migration reaction is triggered with the blue light of 450 nm where is the maximum flavin excitation in BcLOV; and reversed detaching from membrane occurs in the dark. Thus this mechanism could be useful to manageably locate specifically proteins fused with BcLOV4. It creates in this way a kind of compartmentalisation in the bacterial cell that could be used to improve the accuracy of other transcriptional regulators. We used truncated BcLOV4 variant - BcLOV4 ∆1-240 with codon optimization for E.coli. This procedure facilitate protein binding to membrane even in dim light (Fig. 1) [2]. Codon optimization of the nucleotide sequence for efficient gene expression in E.coli was performed using GENEWIZ and verified against a triplet frequency table obtained from the Codon Usage Database.
During our project, we confirmed with a computer simulation that membrane localization of shortened BcLOV4 without N-terminal G-protein signalling (RGS) domain is mediated by a polybasic amphipathic helix located right after LOV domain (Fig. 2).
The molecular dynamics of BcLOV4 was calculated using a set of CHARMM force fields. The BcLOV4 molecular dynamics values were obtained in GROMACS program. We simulated the binding of the BcLOV4 alpha helix to the lipid membranes of E. coli (Epot = -2.3×e+6 kJ×mol-1) and Spirulina B-256 (Epot = -2.1×e+6 kJ×mol-1). E. coli accumulates two major membrane phospholipids: phosphatidylethanolamine, phosphatidylglycerol, while the spirulina membrane is consisted of glycolipids: monogalactosyl diacylglycerolipids, digalactosyl diacylglycerolipids, and sulfoquinovosyl diacylglycerolipids.
Since there are no models of these membranes in the CHARMM-GUI database, we decided to create dipalmitoyl phosphatidylethanolamine (C16:0/16:0) membrane model for E. coli and dipalmitoyl glycerol (C16:0/16:0) membrane model for spirulina in CHARMM-GUI.
The results demonstrate that membrane localization of RGS-truncated BcLOV4 protein is mediated by a polybasic amphipathic helix after the LOV domain (RMSD < 0.5 nm for 100 ps). We also showed that the reversible electrostatic interaction dependent on the anionic content of the membrane without preference for a specific group.
Codon optimization of the nucleotide sequence for efficient gene expression in Arthrospira platensis was performed using GENEWIZ and verified against a triplet frequency table obtained from the Codon Usage Database.
1. Single-Component Optogenetic Tools for Inducible RhoA GTPase Signaling. Adv Biol (Weinh), 2021
2. Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proc Natl Acad Sci USA, 2018
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 354
Illegal PstI site found at 598 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 354
Illegal PstI site found at 598 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 354
Illegal PstI site found at 598 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 354
Illegal PstI site found at 598 - 1000COMPATIBLE WITH RFC[1000]
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