Winter, Kerstin ; Born, Johannes ; Pfeifer, Felicitas (2018)
Interaction of Haloarchaeal Gas Vesicle Proteins Determined by Split-GFP.
In: Frontiers in microbiology, 9
Artikel, Bibliographie
Dies ist die neueste Version dieses Eintrags.
Kurzbeschreibung (Abstract)
Several extremely halophilic archaea produce proteinaceous gas vesicles consisting of a gas-permeable protein wall constituted mainly by the gas vesicle proteins GvpA and GvpC. Eight additional accessory Gvp are involved in gas vesicle formation and might assist the assembly of this structure. Investigating interactions of halophilic proteins requires a method functioning at 2.5-5 M salt, and the split-GFP method was tested for this application. The two fragments NGFP and CGFP do not assemble a fluorescent GFP protein when produced , but they assemble a fluorescent GFP when fused to interacting proteins. To adapt the method to high salt, we used the genes encoding two fragments of the salt-stable mGFP2 to construct four vector plasmids that allow an N- or C-terminal fusion to the two proteins of interest. To avoid a hindrance in the assembly of mGFP2, the fusion included a linker of 15 or 19 amino acids. The small gas vesicle accessory protein GvpM and its interaction partners GvpH, GvpJ, and GvpL were investigated by split-GFP. Eight different combinations were studied in each case, and fluorescent transformants indicative of an interaction were observed. We also determined that GvpF interacts with GvpM and uncovered the location of the interaction site of each of these proteins in GvpM. GvpL mainly interacted with the N-terminal 25-amino acid fragment of GvpM, whereas the other three proteins bound predominately to the C-terminal portion. Overall, the split-GFP method is suitable to investigate the interaction of two proteins in haloarchaeal cells. In future experiments, we will study the interactions of the remaining Gvps and determine whether some or all of these accessory Gvp proteins form (a) protein complex(es) during early stages of the assembly of the gas vesicle wall.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2018 |
| Autor(en): | Winter, Kerstin ; Born, Johannes ; Pfeifer, Felicitas |
| Art des Eintrags: | Bibliographie |
| Titel: | Interaction of Haloarchaeal Gas Vesicle Proteins Determined by Split-GFP. |
| Sprache: | Englisch |
| Publikationsjahr: | 17 August 2018 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Frontiers in microbiology |
| Jahrgang/Volume einer Zeitschrift: | 9 |
| Zugehörige Links: | |
| Kurzbeschreibung (Abstract): | Several extremely halophilic archaea produce proteinaceous gas vesicles consisting of a gas-permeable protein wall constituted mainly by the gas vesicle proteins GvpA and GvpC. Eight additional accessory Gvp are involved in gas vesicle formation and might assist the assembly of this structure. Investigating interactions of halophilic proteins requires a method functioning at 2.5-5 M salt, and the split-GFP method was tested for this application. The two fragments NGFP and CGFP do not assemble a fluorescent GFP protein when produced , but they assemble a fluorescent GFP when fused to interacting proteins. To adapt the method to high salt, we used the genes encoding two fragments of the salt-stable mGFP2 to construct four vector plasmids that allow an N- or C-terminal fusion to the two proteins of interest. To avoid a hindrance in the assembly of mGFP2, the fusion included a linker of 15 or 19 amino acids. The small gas vesicle accessory protein GvpM and its interaction partners GvpH, GvpJ, and GvpL were investigated by split-GFP. Eight different combinations were studied in each case, and fluorescent transformants indicative of an interaction were observed. We also determined that GvpF interacts with GvpM and uncovered the location of the interaction site of each of these proteins in GvpM. GvpL mainly interacted with the N-terminal 25-amino acid fragment of GvpM, whereas the other three proteins bound predominately to the C-terminal portion. Overall, the split-GFP method is suitable to investigate the interaction of two proteins in haloarchaeal cells. In future experiments, we will study the interactions of the remaining Gvps and determine whether some or all of these accessory Gvp proteins form (a) protein complex(es) during early stages of the assembly of the gas vesicle wall. |
| ID-Nummer: | pmid:30174663 |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Microbiology and Archaea |
| Hinterlegungsdatum: | 06 Sep 2018 07:59 |
| Letzte Änderung: | 03 Jul 2024 02:30 |
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| Suche nach Titel in: | TUfind oder in Google |
Verfügbare Versionen dieses Eintrags
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Interaction of Haloarchaeal Gas Vesicle Proteins Determined by Split-GFP. (deposited 04 Nov 2018 20:55)
- Interaction of Haloarchaeal Gas Vesicle Proteins Determined by Split-GFP. (deposited 06 Sep 2018 07:59) [Gegenwärtig angezeigt]
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