Molecular Architecture and Electrostatic Properties of a Bacterial Porin

Weiss, M.S.; Abele, Ulrich; Weckesser, Jürgen; Welte, Wolfram; Schiltz, Emile; Schulz, Georg E.

doi:10.1126/science.1721242

Cited by 687 publications

(406 citation statements)

References 28 publications

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“…Recently, however, nonhelical channel-forming polypeptides have also been described, including the bacterialpore protein porin (Weiss et al, 1991) and the human tumor necrosis factor (TNF)-a Kagan et al, 1992). Porin consists of trimers of &barrels that form pores.…”

Section: Methodsmentioning

confidence: 99%

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

1994

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Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions.Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the P0PG:POPC ratio. Vesicles (300 pM POPG) readily aggregated at HNP-2 concentrations above 1 pM, but no mixing of vesicle contents could be detected for concentrations as high as 2 pM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 pM POPG vesicles, 50% of entrapped solute is released by 0.7 p M HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (-400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.

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Section: Methodsmentioning

confidence: 99%

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

1994

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“…These results are in agreement with the previously established predominant reactivity of free surface accessible lysine residues toward amino acylation (Glocker et al, 1994). Three other lysine residues (Lys-138, Lys-169, Lys-230) on the outer surface of the barrel are not succinylated, which can be explained by shielding due to surrounding detergent molecules (Weiss et al, 1991a). The low reactivity of the Lys-3 residue at the inner channel surface toward succinylation can be explained by shielding due to the Ser-5 and Thr-30 residues, and by a local conformational change that may occur on succinylation of the adjacent N-terminal (Glu-1)-amino group Glocker et al, 1994).…”

Section: Average B-factors (A*)mentioning

confidence: 99%

“…1). The permeation of hydrated ions through this constriction loop may require transient stripping of part of their hydration shells (Weiss et al, 1991a).…”

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confidence: 99%

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X‐ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from rhodobacter capsulatus: Implications for ion selectivity and single‐channel conductance

et al. 1996

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The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysinet-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal a-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.

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“…improve the signal-to-noise ratio, the conspicuous accumulation of aromatic residues in flanking positions (Kinemage 3; Weiss et al, 1991;Cowan et al, 1992) was exploited. A modified 0-side-hydrophobicity, H;, was defined by assigning an arbitrarily increased "hydrophobicity" index of 1.6 to aromatic residues (instead of the consensus indices ranging from -0.4 to 1.19 for these residues) when encountered in positions i -2 or i + 4.…”

mentioning

confidence: 99%

Prediction of membrane‐spanning β‐strands and its application to maltoporin

Schirmer¹,

Cowan²

1993

Protein Science

120

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Integral membrane proteins often exhibit long hydrophobic stretches in their amino acid sequence that indicate membrane-spanning a-helices and therefore provide useful topological information. Accordingly, the algorithm of Kyte and Doolittle (1982), which determines the mean hydrophobicity within a sliding window, is routinely applied to new membrane protein sequences to identify transmembrane helices. There is, however, another class of membrane proteins, exemplified by bacterial porins, that are folded predominantly into @-pleated sheet structures and show an overall hydrophobicity similar to soluble proteins.The recent crystal structure elucidations of matrix porin and phosphoporin from Escherichia coli (Cowan et al., 1992) and the unrelated porin from R . capsulatus (Weiss & Schulz, 1992) prompted us to reevaluate existing prediction methods for transmembrane @-strands. All of these porins are folded into 16-stranded antiparallel @-barrels (Kinemage 1) that are associated to homotrimers. The parts of the 0-strands that are in contact with the hydrophobic core of the membrane are 7-9 residues in length with every second (i.e., external) residue being hydrophobic (Kinemage 2). The intervening, internal residues show no clear pattern and thus have no predictive value. These residues are found to be hydrophilic when being part of the pore lumen or hydrophobic when buried by the internal loop L3 (Cowan et al., 1992).The mean hydrophobicity of one side of a putative @-strand (0-side-hydrophobicity, H,) is obtained by averaging the hydrophobic indices h (Eisenberg et al., 1984) of every second residue within a sliding window (Vogel & Jahnig, 1986). A window width of 4 was used so that

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Molecular Architecture and Electrostatic Properties of a Bacterial Porin

Cited by 687 publications

References 28 publications

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

X‐ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from rhodobacter capsulatus: Implications for ion selectivity and single‐channel conductance

Prediction of membrane‐spanning β‐strands and its application to maltoporin

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