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1.  Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract 
A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity.
doi:10.1021/la047732f
PMCID: PMC3608253  PMID: 15697298
2.  Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na+ channels 
Biochimica et Biophysica Acta  2011;1818(3):443-447.
The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na+ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na+ current (INa). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in INa. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4 min), there is little effect on INa. However, with longer exposures (4–8 min), there is a large decrease in INa, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na+ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.
doi:10.1016/j.bbamem.2011.11.031
PMCID: PMC3372909  PMID: 22155680
hNav1.5; hNav1.5-C373Y; hNav1.4; Methanethiosulfonate (MTSEA); Slow inactivation; Fast inactivation
3.  Crystallization of the hydantoin transporter Mhp1 from Microbacterium liquefaciens  
Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.
The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R merge of 0.12. Further experimental phasing methods are under way.
doi:10.1107/S1744309108036920
PMCID: PMC2593711  PMID: 19052379
transporters; nucleobase:cation symporter 1 family; membrane proteins; hydantoins
4.  Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter 
Science (New York, N.Y.)  2008;322(5902):709-713.
The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
doi:10.1126/science.1164440
PMCID: PMC2885439  PMID: 18927357
5.  Adult bronchiolitis and parainfluenza type 2 
Postgraduate Medical Journal  1980;56(661):787-788.
A case of acute bronchiolitis in association with parainfluenza type 2 infection is reported in a previously asymptomatic young adult with evidence of partially reversible severe airways obstruction.
PMCID: PMC2426058  PMID: 6267572

Results 1-5 (5)