Cloning and expression of multiple integral membrane proteins from <i>Mycobacterium tuberculosis</i> in <i>Escherichia coli</i>

Korepanova, Alla; Gao, Fei; Hua, Yuanzi; Qin, Huajun; Nakamoto, Robert K.; Cross, Timothy A.

doi:10.1110/ps.041022305

Cited by 91 publications

(65 citation statements)

References 45 publications

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“…S1). The structured portions in the N-terminal region (residues [19][20][21][22][23][24][25][26][27] and in the loop (residues 56-67) have remarkably uniform chemical shifts between 70 and 85 ppm and dipolar couplings between 4.3 and 5.5 kHz, with only a couple of outliers for each parameter. The immediate conclusion is these residues form α-helices or β-strands, with the peptide N-H bonds nearly parallel to the bilayer surface, such as an amphipathic helix or a β-sheet in the plane of the bilayer interface.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, watersoluble domains of other Mtb membrane proteins have been characterized such as those from PknB and FtsX (21)(22)(23)(24). Although X-ray crystallographers have focused on large membrane proteins, the majority of the 1,162 ORFs of the Mtb genome code for small helical membrane proteins containing one to three TM helices with <40-kDa molecular weight (25). Structurefunction studies of these small membrane proteins are essential for understanding Mtb cell division and other cellular processes.…”

Section: Significancementioning

confidence: 99%

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Structure of CrgA, a cell division structural and regulatory protein fromMycobacterium tuberculosis, in lipid bilayers

Das

Dai

Hung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines.

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Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Structure of CrgA, a cell division structural and regulatory protein fromMycobacterium tuberculosis, in lipid bilayers

Das

Dai

Hung

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…We discovered that the MmsF protein and the homologs were overexpressed in a highly soluble form in E. coli, despite being predicted to have three TMS helices. This finding is a very unusual result, with only a limited number of membrane proteins capable of even existing in a stable form in aqueous environments without the addition of detergent or lipid (24). Magnetosome-associated proteins that have been previously characterized, such as Mms6 and, recently, Mms13 (MamC), are also predicted to span the MM.…”

Section: Discussionmentioning

confidence: 83%

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

Rawlings

Bramble

Walker

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Magnetotactic bacteria synthesize highly uniform intracellular magnetite nanoparticles through the action of several key biomineralization proteins. These proteins are present in a unique lipidbound organelle (the magnetosome) that functions as a nanosized reactor in which the particle is formed. A master regulator protein of nanoparticle formation, magnetosome membrane specific F (MmsF), was recently discovered. This predicted integral membrane protein is essential for controlling the monodispersity of the nanoparticles in Magnetospirillum magneticum strain AMB-1. Two MmsF homologs sharing over 60% sequence identity, but showing no apparent impact on particle formation, were also identified in the same organism. We have cloned, expressed, and used these three purified proteins as additives in synthetic magnetite precipitation reactions. Remarkably, these predominantly α-helical membrane spanning proteins are unusually highly stable and water-soluble because they self-assemble into spherical aggregates with an average diameter of 36 nm. The MmsF assembly appears to be responsible for a profound level of control over particle size and iron oxide (magnetite) homogeneity in chemical precipitation reactions, consistent with its indicated role in vivo. The assemblies of its two homologous proteins produce imprecise various iron oxide materials, which is a striking difference for proteins that are so similar to MmsF both in sequence and hierarchical structure. These findings show MmsF is a significant, previously undiscovered, protein additive for precision magnetite nanoparticle production. Furthermore, the self-assembly of these proteins into discrete, soluble, and functional "proteinosome" structures could lead to advances in fields ranging from membrane protein production to drug delivery applications.MmsF | proteinosome | magnetite | magnetosome | in vitro precipitation M agnetic nanoparticles (MNPs) represent an area of intense research due to their diverse and pertinent applications across a range of disciplines and industries. Applications for MNPs include biomedical diagnostics and therapies (1-3), such as MRI contrast reagents, tumor hyperthermia treatments, and magnetically targeted drug delivery, as well as data storage (4) and biotechnology. However, specific magnetic and physical properties of MNPs are critical to the success of each application, with specific size and morphology (with a narrow distribution) being essential considerations. Pure magnetite MNP synthesis under ambient conditions is notoriously difficult to control, with simple precipitations often resulting in a mixture of differently sized and shaped particles with other iron oxide contaminants. This situation can be improved somewhat by using more extreme processes, such as high-temperature incubations or capping surfactants, which favor certain MNP types (5, 6). However, the use of toxic or organic reagents and extreme conditions comes with a high energy and monetary cost, and can limit the biocompatibility of the MNPs for subsequent app...

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“…This problem is illustrated in a study by Korepanova et al wherein the Mycobacterium tuberculosis (MTb) a-helical membrane proteome was expression-tested in various Escherichia coli strains. 1 Out of the 105 membrane protein targets tested, only 37 were over-expressed sufficiently to be detectable by Coomassie staining. Only nine of those 37, all less than 16 kD, were expressed to the membrane fraction and presumably correctly folded.…”

Section: Introductionmentioning

confidence: 99%

Genetic selection system for improving recombinant membrane protein expression in E. coli

et al. 2009

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A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

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Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli

Cited by 91 publications

References 45 publications

Structure of CrgA, a cell division structural and regulatory protein fromMycobacterium tuberculosis, in lipid bilayers

Structure of CrgA, a cell division structural and regulatory protein fromMycobacterium tuberculosis, in lipid bilayers

Self-assembled MmsF proteinosomes control magnetite nanoparticle formation in vitro

Genetic selection system for improving recombinant membrane protein expression in E. coli

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