Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3.

Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating.

Stabilising and destabilising modifications of cysteines in the E. coli outer membrane porin protein OmpC.

Three sulfhydryl labels were used to modify two mutated sites, R37C and R74C in the eyelet of the outer membrane porin OmpC. Modification of R37C with the neutral groups Aldrithiol and bimane increases thermal stability but the negatively charged iodoacetate causes a decrease in thermal stability. The effects of substitution at R74C were less significant. Bimane labelling increases the voltage sensitivity and decreases the single channel conductance at R37C asymmetrically with smaller channels being recorded at cis negative voltages. Negatively charged acetate does not affect the voltage gating.

Voltage gating is a fundamental feature of porin and toxin beta-barrel membrane channels.

Beta-barrel pores are found in outer membrane porins of gram-negative bacteria, bacterial toxins and mitochondrial channels. Apart from the beta-barrel the three groups show no close sequence or structural homology but these pores exhibit symmetrical voltage gating when reconstituted into planar lipid bilayers. The structures of several of these are known and many site-directed mutants have been examined. As a result it seems evident that the gating is a common characteristic of these unrelated large pores and is not generated by specialised structures in the pore lumen.

Displacement of OmpF loop 3 is not required for the membrane translocation of colicins N and A in vivo.

The pore-forming colicins N and A require the porin, OmpF, in order to translocate across the outer membrane of Escherichia coli. We investigated the hypothesis that in vivo, colicins N and A may traverse the outer membrane through the OmpF channel. In order to accommodate a polypeptide in the pore, the mid-channel constriction loop of OmpF, L3, would need to undergo a conformational change. We used five OmpF cystine mutants, which fix L3 in the conformation determined by X-ray crystallography, to investigate L3 movement during colicin activity in vivo. Sensitivity to colicins N and A of E. coli cells expressing these OmpF cystine mutants was determined using cell survival and in vivo potassium efflux and fluorescence assays. Results indicate that gross movement of L3 is not required for colicin N or A activity and that neither of these colicins crosses the outer membrane of E. coli through the lumen of the OmpF pore.

Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

Discovery of critical Tol A-binding residues in the bactericidal toxin colicin N: a biophysical approach.

Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After first binding to outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal regions. Finally, the toxic C-terminal region is inserted into or across the cytoplasmic membrane. We have measured the binding of colicin N to TolA by isothermal titration microcalorimetry (ITC) and tryptophan fluorescence. The isolated N-terminal domain exhibits a higher affinity for TolA (Kd = 1 microM) than does the whole colicin (18 microM), and similar behaviour has been observed when the N-terminal domain of the g3p protein of the bacteriophage fd, which also binds TolA, is examined in isolation and in situ. This may indicate a similar mechanism in which a cryptic TolA binding site is revealed after primary receptor binding. The isolated colicin N N-terminal domain appears to be unstructured in circular dichroism and fluorescence studies. We have used mutagenesis and ITC to characterize the TolA binding site and have shown it to be of a different sequence and much further from the N-terminus than previously thought.

Steady-state free precession magnetic resonance imaging of the heart: comparison with segmented k-space gradient-echo imaging.

Steady-state free precession imaging is a promising technique for cardiac magnetic resonance imaging (MRI), as it provides improved blood/myocardial contrast in shorter acquisition times compared with conventional gradient-echo acquisition. The better contrast could improve observer agreement and automatic detection of cardiac contours for volumetric assessment of the ventricles, but measurements might differ from those obtained using conventional methods. We compared volumetric measurements, observer variabilities, and automatic contour detection between a steady-state free precession imaging sequence (BFFE = balanced fast field echo) and segmented k-space gradient-echo acquisition (TFE = turbo field echo) in 41 subjects. With BFFE, significantly higher end-diastolic and end-systolic volumes and lower wall thickness, ventricular mass, ejection fraction, and wall motion were observed (P < 0.0001), while interobserver variabilities were lower and automatic contour detection of endocardial contours was more successful. We conclude that the improved image quality of BFFE reduces the observer-dependence of volumetric measurements of the left ventricle (LV) but results in significantly different values in comparison to TFE measurements.

Colicin pore-forming domains bind to Escherichia coli trimeric porins.

Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin-P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin-porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.