Structure analysis of the membrane protein TatCd from the Tat system of B. subtilis by circular dichroism |
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| Authors: | Olga V. Nolandt Siegmar Roth Anne S. Ulrich |
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| Affiliation: | a Karlsruhe Institute of Technology (KIT), Institut für Biologische Grenzflächen (IBG-2), Forschungszentrum Karlsruhe, P. O. B. 3640, D-76021 Karlsruhe, Germany
b Karlsruhe Institute of Technology (KIT), Institut für Organische Chemie, Universität Karlsruhe (TH), Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany |
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| Abstract: | The twin arginine translocation (Tat) system can transport fully folded proteins, including their cofactors, across bacterial and thylakoid membranes. The Tat system of Bacillus subtilis that serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatAd and TatCd. The larger component TatCd has a molecular weight of 28 kDa and several membrane-spanning segments. This protein has been expressed in Escherichia coli and purified in sufficient amounts for structure analysis by circular dichroism (CD) and NMR spectroscopy. TatCd was reconstituted in detergent micelles and in lipid bilayers for CD analysis in solution and in macroscopically oriented samples, to examine the stability of the protein. Suitable protocols and model membrane systems have been established, by which TatCd maintains the level of helicity close to theoretically predicted, and its transmembrane alignment could been verified. |
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| Keywords: | Tat, twin arginine translocation CD, circular dichroism OCD, oriented circular dichroism DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPG, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine DPC, n-dodecylphosphocholine LPPG, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] LMPC, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine β-OG, β-octyl-glucoside β-NG, β-nonyl-glucoside DDM, β-dodecylmaltoside NLS, N-lauroylsarcosine SDS, sodium dodecylsulfate IPTG, isopropyl-β- smallcaps" >d-1-thiogalactopyranosid LB, Luria-Bertani MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry HSQC, heteronuclear single quantum coherence |
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