Difference between revisions of "Part:BBa K4044003"
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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). | 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. | 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 ( | + | 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. |
− | [[File:T--LMSU--Modeling BcLOV4 membr.png|400px|thumb|center|BcLOV4 attached to membrane by amphipathic helix]] | + | |
− | + | [[File:T--LMSU--Modeling BcLOV4 membr.png|400px|thumb|center|Fig. 1 BcLOV4 attached to membrane by amphipathic helix]] | |
− | During our project, we confirm with a computer simulation that membrane localisation of shortened BcLOV4 without N-terminal G-protein signalling (RGS) domain is mediated by a polybasic amphipathic helix located right after LOV domain. We also showed that this reversible electrostatic interaction is non-selective among anionic phospholipids and depends only on the content of anions in a membrane ( | + | |
− | + | During our project, we confirm with a computer simulation that membrane localisation of shortened BcLOV4 without N-terminal G-protein signalling (RGS) domain is mediated by a polybasic amphipathic helix located right after LOV domain. We also showed that this reversible electrostatic interaction is non-selective among anionic phospholipids and depends only on the content of anions in a membrane (Fig. 2). | |
+ | |||
+ | [[File:T--LMSU--Modeling BcLOV4 membr-E-coli-gif.gif|400px|thumb|center|Fig. 2 Molecular dynamics of BcLOV4 attached to dipalmitoyl phosphatidylethanolamine]] | ||
+ | |||
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. | 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. | ||
Revision as of 16:41, 20 October 2021
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 confirm with a computer simulation that membrane localisation of shortened BcLOV4 without N-terminal G-protein signalling (RGS) domain is mediated by a polybasic amphipathic helix located right after LOV domain. We also showed that this reversible electrostatic interaction is non-selective among anionic phospholipids and depends only on the content of anions in a membrane (Fig. 2).
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]