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Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into(More)
Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport [Wrubel, W., Stochaj, U., et al. (1990) J. Bacteriol. 172, 5374-5381]. Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity(More)
The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three(More)
The interactions between the mitochondrial cytochrome bc(1) complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc(1) complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc(1) complex(More)
The Sonic hedgehog (SHh) pathway plays a critical role in normal embryogenesis and carcinogenesis, but its function in cancer cells treated with 5-fluorouracil (5-FU) remains unknown. We examined the expression of a subset of SHh signaling pathway genes, including SHh, SMO, PTC1, Su(Fu) and HIP in human hepatocellular carcinoma (HCC) cell lines, Hep3B and(More)
The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227,(More)
Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity. The intrinsic fluorescence(More)
The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of(More)
Interactions between Rhodobacter sphaeroides cytochrome bc1 complex (Rsbc1) and soluble cytosolic proteins were studied by a precipitation pull-down technique. After being purified, detergent-dispersed Rsbc1 complex was incubated with soluble cytosolic fraction and then dialyzed in the absence of detergent; the interacting proteins were coprecipitated with(More)
The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet(More)
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