Abstract
MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.
Highlights
Firmed by differential scanning calorimetry [4]
Since wild-type MspA does not contain any cysteine residues, 52 out of 184 amino acids of the mature protein were directly replaced by cysteines using site-directed mutagenesis (Fig. 1). The positions for these mutations were selected before the crystal structure of MspA was published [16] using the following criteria. (i) Putative extracellular loops were predicted by a neural network specialized for outer membrane proteins [28] and by an algorithm specialized for prediction of membrane-spanning -strands [29, 30] and were preferably targeted to gain topological information. (ii) Charged amino acids are often located in extracellular loops and may play functional roles by determining the charge selectivity of MspA
To examine whether the MspA cysteine mutants were expressed in the outer membrane (OM) of M. smegmatis, a selective extraction procedure was employed that yields predominantly MspA when whole cells of M. smegmatis are heated with 0.5% octyl-POE to 100 °C [26]
Summary
Chemicals and Enzymes—Chemicals were of the highest purity available from Merck, Roth, or Sigma. Biotinylation of Native MspA Cysteine Mutants in Vitro and in Vivo— For biotinylation of the MspA cysteine mutants in vitro and in vivo cells were grown to an optical density A600 of 0.8, harvested by centrifugation, and washed three times with 2 ml of ice-cold PBS (100 mM NaH2PO4/ Na2HPO4, 0.1 mM Na2EDTA, 150 mM NaCl, 0.05% (m/v) Tween 80, pH 7). After washing with PBS and resuspending in POP05 buffer (300 mM NaH2PO4/Na2HPO4, 0.3 mM Na2EDTA, 150 mM NaCl, 0.5% (m/v) N-octyl-POE) to a cell density of 10 mg/100 l, the suspension was boiled under stirring for 30 min in a water bath. The supernatant contained the MspA mutant protein Both supernatants, obtained after biotinylation of the MspA mutant protein in vitro or within M. smegmatis cells, were further analyzed for protein content by gel electrophoresis and for the amount of biotin label by Western blot experiments. The mutant E139C was chosen as the positive control based on this analysis
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