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 [2].
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.
The molecular dynamics of BcLOV4 was calculated using a set of CHARMM force fields. The BcLOV4 molecular dynamics values were obtained in GROMACS program. The result is reliable if Epot is negative, and on the order of 106-107 for proteins in water, depending on the system size. During the energy minimization phase, the system maximum force should not exceed 1000 kJ mol-1 nm-1. We simulated the binding of the BcLOV4 alpha helix to the lipid membranes of E. coli (Epot = -2.3×106 kJ mol-1) and Arthrospira platensis IPPAS B-256 (Epot = -2.1×106 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 E. coli 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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