TetL Tetracycline Efflux Protein from <i>Bacillus subtilis </i>Is a Dimer in the Membrane and in Detergent Solution

Safferling, Markus; Griffith, Heather; Jin, Jiashun; Sharp, Josh S.; Jesus, Magdia De; Ng, Caroline L.; Krulwich, Terry A.; Wang, Da-Neng

doi:10.1021/bi035173q

Cited by 23 publications

(20 citation statements)

References 43 publications

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“…TetL from Bacillus subtilis (BsTetL) is an integral membrane protein that induces tetracycline resistance by exporting tetracycline from the bacterial cytoplasm (45). TetL is a member of the major facilitator superfamily, although the TetL subfamily has an atypical number of transmembrane helices (number of helices ¼ 14).…”

Section: Resultsmentioning

confidence: 99%

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Rapid Bioinformatic Identification of Thermostabilizing Mutations

Sauer

Karpowich

Song

et al. 2015

Biophysical Journal

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Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.

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Section: Resultsmentioning

confidence: 99%

“…The TetL-GFP fusion had an expression level of~0.75 mg/L, which is slightly lower than that of the His-tagged construct (45). Lysates were split into two aliquots and incubated at either 4 C or 42 C, and then both samples were examined by analytical SEC with fluorescence detection (41).…”

Section: Screening Of Bstetl Mutants For Thermostabilitymentioning

confidence: 99%

Rapid Bioinformatic Identification of Thermostabilizing Mutations

Sauer

Karpowich

Song

et al. 2015

Biophysical Journal

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“…Deletion analyses of the S. aureus [Hiramatsu et al, 1998] and B. subtilis [Ito et al, 2000] operons demonstrated that all the mrp gene products are required for the maximal Mrp-associated Na + resistance. These findings indicate that the Mrp antiporters probably function as hetero-oligomeric complexes, in contrast to most other prokaryotic secondary monovalent cation/proton exchangers which are single membrane polypeptides or homo-oligomers [Gerchman et al, 2001;Safferling et al, 2003].…”

Section: Introductionmentioning

confidence: 88%

The <i>Vibrio cholerae</i> Mrp System: Cation/Proton Antiport Properties and Enhancement of Bile Salt Resistance in a Heterologous Host

Dzioba-Winogrodzki

Winogrodzki²,

Krulwich³

et al. 2008

Microb Physiol

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The mrp operon from Vibrio cholerae encoding a putative multisubunit Na⁺/H⁺ antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na⁺ and Li⁺ as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li⁺, Na⁺, and K⁺ ions in pH-dependent manner with maximal activity at pH 9.0–9.5. Exchange was electrogenic (more than one H⁺ translocated per cation moved in opposite direction). The apparent K_m at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li⁺, Na⁺, and K⁺, respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.

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“…There are integral membrane proteins that are purified as monomers but are believed to function as dimers or oligomers. For example, a tetracycline-divalent cation efflux protein TetL was suggested to exist as an oligomer rather than a monomer in the membrane based on SDS-PAGE and Western blot analysis of membrane samples (43). However, exploring membrane-induced oligomerization of peripheral membrane proteins, particularly where such aggregation might not be necessary for enzymatic activity but rather enhance catalysis, is very difficult.…”

Section: Discussionmentioning

confidence: 99%

Modulation of Bacillus thuringiensis Phosphatidylinositol-specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

Shi

Shao

Zhang

et al. 2009

Journal of Biological Chemistry

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Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/ Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/ Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.

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TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution

Cited by 23 publications

References 43 publications

Rapid Bioinformatic Identification of Thermostabilizing Mutations

Rapid Bioinformatic Identification of Thermostabilizing Mutations

The <i>Vibrio cholerae</i> Mrp System: Cation/Proton Antiport Properties and Enhancement of Bile Salt Resistance in a Heterologous Host

Modulation of Bacillus thuringiensis Phosphatidylinositol-specific Phospholipase C Activity by Mutations in the Putative Dimerization Interface

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