Mutational Analyses Define Helix Organization and Key Residues of a Bacterial Membrane Energy-transducing Complex (original) (raw)
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Ion channel activities in the Escherichia coli outer membrane
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.
The Journal of Biological Chemistry, 2009
The TolQRA proteins of Escherichia coli form an inner membrane complex involved in the maintenance of the outer membrane stability and in the late stages of cell division. The TolQR complex uses the proton motive force to regulate TolA conformation and its interaction with the outer membrane Pal lipoprotein. It has been proposed that an ion channel forms at the TolQR transmembrane helix (TMH) interface. This complex assembles with a minimal TolQ:TolR ratio of 4 -6:2 and therefore involves 14 -20 TMHs. To define the organization of the transmembrane helices in the membrane within the TolQR complex, we initiated a cysteine scanning study. In this study, we report results for the systematic replacement of each residue of the TolR TMH. Phenotypic analyses first showed that most of the mutants are functional. Three mutants, TolR L22C, D23C, and V24C, were shown to affect TolQR functioning. Disulfide bond complex formation further showed that two TolR anchors are close enough to interact. Two substitutions, L22C and V24C, form high level of dimers, suggesting that the TolR helix rotates as molecular gears between these two positions and that disulfide bond formation between these residues blocked the rotary motion. Mutations of critical residues located within the TolQ TMH2 and TMH3 and the TolR TMH and proposed to form the ion pathway prevent rotation between these two residues. TolR anchors may form molecular gears that oscillate in response to proton motive force to regulate channel activity.
Journal of Biological Chemistry, 2013
Background: Proteins generally require the YidC and SecYEG machineries for membrane insertion. Results: The specific features of membrane protein sequences determine YidC/SecYEG requirements for insertion. Conclusion: The charge composition of membrane proteins determines the YidC and Sec translocase requirements in E. coli. Significance: Translocase requirements for membrane insertion are determined by the charge composition of membrane proteins. We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidConly, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.
Research in Microbiology, 2001
The outer membrane of Gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities. 2001 Éditions scientifiques et médicales Elsevier SAS
Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane
Biophysical Journal, 2009
We demonstrate the energy dependence of the motion of a porin, the l-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual l-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the l-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the l-receptor significantly decreased. Since the l-receptor is closely linked to the peptidoglycan layer, we propose that l-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.
The structure and mechanism of the TolC outer membrane transport protein
2004
Gram-negative bacteria have evolved specialized multicomponent systems that transport molecules from the cytoplasm to the extracellular environment in energydependent processes. Central to one of these systems is the TolC family of outer membrane proteins. TolC of Escherichia coli is a very versatile channel that can interact with a wide range of inner membrane, energydriven pumps to export compounds ranging from small antibiotic molecules to large toxic proteins. Thus TolC and its associated partner proteins confer invasive virulence and drug resistance to Gram-negative bacterial pathogens. However TolC is also a source of vulnerability, as it is a conduit for the uptake of bactericidal proteins known as colicins. Recently the crystal structures have been reported of TolC and its inner-membrane partner protein AcrB, a proton antiporter. The mechanisms of colicin uptake via TolC have also been extensively studied and the structures of some of the implicated protein components have been determined. In this review we focus on the current understanding of the structure and function relationships in TolC-mediated transport systems.
Journal of bacteriology, 1993
The TolQ and TolR proteins of Escherichia coli are required for the uptake of group A colicins and for infection by filamentous phages. Their topology in the cytoplasmic membrane was determined by cleavage with aminopeptidase K, proteinase K, and trypsin in spheroplasts and cell lysates. From the results obtained, it is proposed that the N terminus of TolQ is located in the periplasm and that it contains three transmembrane segments (residues 9 to 36, 127 to 159, and 162 to 191), a small periplasmic loop, and two large portions in the cytoplasm. The N terminus of TolR is located in the cytoplasm and is followed by a transmembrane segment (residues 21 to 40), and the remainder of the protein is located in the periplasm. A tolQ mutant, which rendered cells resistant to group A colicins and sensitive to cholate, had alanine 13 replaced by glycine and was lacking serine 14 in the first transmembrane segment. The membrane topologies of TolQ and TolR are similar to those proposed for ExbB...
The EMBO Journal, 1986
The amino acid distribution in membrane spanning segments and connecting loops in bacterial inner membrane proteins was analysed. The basic residues Arg and Lys are four times less prevalent in periplasmic as compared to cytosolic connecting loops, whereas no comparable effect is observed for the acidic residues Asp and Glu. Also, Pro is shown to be tolerated to a much larger extent in membrane spanning segments with their N-terminus pointing towards the cytosol than in those with the opposite orientation. The significance of these findings with regard to the mechanism of biogenesis of bacterial inner membrane proteins is discussed.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2003
The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B 12 across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque. D