Difference between revisions of "Part:BBa K4044003"
Alexandr56 (Talk | contribs) |
Alexandr56 (Talk | contribs) |
||
(13 intermediate revisions by the same user not shown) | |||
Line 3: | Line 3: | ||
<partinfo>BBa_K4044003 short</partinfo> | <partinfo>BBa_K4044003 short</partinfo> | ||
− | 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 [2]. |
− | [[File:T--LMSU--Modeling BcLOV4 membr.png|400px|thumb|center| | + | [[File:T--LMSU--Modeling BcLOV4 membr.png|400px|thumb|center|BcLOV4 attached to membrane by amphipathic helix]] |
− | + | 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. | |
− | [[File:T--LMSU--Modeling BcLOV4 membr-E-coli-gif.gif|400px|thumb|center| | + | [[File:T--LMSU--Modeling BcLOV4 membr-E-coli-gif.gif|400px|thumb|center|Molecular dynamics of BcLOV4 attached to dipalmitoyl phosphatidylethanolamine (C16:0/16:0) membrane (''E. coli'' membrane model)]] |
+ | [[File:T--LMSU--Modeling-BcLOV4-memb-SP.gif|400px|thumb|center|Molecular dynamics of BcLOV4 attached to dipalmitoyl glycerol (C16:0/16:0) membrane (''Arthrospira platensis'' IPPAS B-256 membrane model)]] | ||
− | Codon optimization of the nucleotide sequence for efficient gene expression in | + | 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 10<sup>6</sup>-10<sup>7</sup> for proteins in water, depending on the system size. During the energy minimization phase, the system maximum force should not exceed 1000 kJ mol<sup>-1</sup> nm<sup>-1</sup>. We simulated the binding of the BcLOV4 alpha helix to the lipid membranes of ''E. coli'' (Epot = -2.3×10<sup>6</sup> kJ mol<sup>-1</sup>) and ''Arthrospira platensis'' IPPAS B-256 (Epot = -2.1×10<sup>6</sup> kJ mol<sup>-1</sup>). ''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. | ||
+ | |||
+ | [[File:T--LMSU--Modeling-RMSD-BcLOV4-memb-SP.png|400px|thumb|center|RMSD for BcLOV4 attached to dipalmitoyl phosphatidylethanolamine (C16:0/16:0) membrane (''E. coli'' membrane model)]] | ||
+ | [[File:T--LMSU--Modeling-En-pot-BcLOV4-memb-SP.png|400px|thumb|center|The system's potential energy for BcLOV4 attached to dipalmitoyl phosphatidylethanolamine (C16:0/16:0) membrane (''E. coli'' membrane model)]] | ||
+ | [[File:T--LMSU--Modeling-RMSD-BcLOV4 membr-E-coli.png|400px|thumb|center|RMSD for BcLOV4 attached to dipalmitoyl glycerol (C16:0/16:0) membrane (''Arthrospira platensis'' IPPAS B-256 membrane model)]] | ||
+ | [[File:T--LMSU--Modeling-En-pot-BcLOV4-membr-E-coli.png|400px|thumb|center|The system's potential energy for BcLOV4 attached to dipalmitoyl glycerol (C16:0/16:0) membrane (''Arthrospira platensis'' IPPAS B-256 membrane model)]] | ||
+ | |||
+ | 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 | 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 | 2. Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proc Natl Acad Sci USA, 2018 | ||
Latest revision as of 22:17, 21 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 [2].
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]