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- Engqvist, Martin, 1983, et al.
(författare)
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Directed evolution of gloeobacter violaceus rhodopsin spectral properties
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:1, s. 205-220
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Tidskriftsartikel (refereegranskat)abstract
- Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ± 80 nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
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- Holmner, Åsa, et al.
(författare)
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Blood group antigen recognition by Escherichia coli heat-labile enterotoxin
- 2007
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Ingår i: Journal of Molecular Biology. - London : Elsevier BV. - 0022-2836 .- 1089-8638. ; 371:3, s. 754-764
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Tidskriftsartikel (refereegranskat)abstract
- In a number of bacterial infections, such as Helicobacter pylori, Campylobacter jejuni and Vibrio cholerae infections, a correlation between the severity of disease and blood group phenotype of infected individuals has been observed. In the present investigation, we have studied the molecular basis of this effect for enterotoxigenic Escherichia coli (ETEC) infections. ETEC are non-invasive bacteria, which act through second messenger pathways to cause diarrhea. It has been suggested that the major virulence factor of ETEC from human isolates, i.e. the human heat-labile enterotoxin (hLT), recognizes certain blood group epitopes, although the molecular basis of blood group antigen recognition is unknown. The 2.5 angstrom crystal structure of the receptor-binding B-subunit of hLT in complex with the blood group A antigen analog GalNAc alpha 3(Fuc alpha-2)Gal beta 4(Fuc alpha-3)Glc beta provides evidence of a previously unknown binding site in the native toxin. The structure reveals the molecular interactions underlying blood group antigen recognition and suggests how this protein can discriminate between different blood group epitopes. These results support the previously debated role of hLT in the blood group dependence of ETEC infections. Similar observations regarding the closely related cholera toxin in V. cholera infections are also discussed. (c) 2007 Elsevier Ltd. All rights reserved.
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- Holmner, Åsa, et al.
(författare)
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Crystal Structures Exploring the Origins of the Broader Specificity of Escherichia coli Heat-Labile Enterotoxin Compared to Cholera Toxin.
- 2011
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 406:3, s. 387-402
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Tidskriftsartikel (refereegranskat)abstract
- Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two binding sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.
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- Hsu, D. S., et al.
(författare)
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FLOW LINEAR DICHROISM AND ELECTRON-MICROSCOPIC ANALYSIS OF PROTEIN-DNA COMPLEXES OF A MUTANT UVRB PROTEIN THAT BINDS TO BUT CANNOT KINK DNA
- 1994
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 241:5, s. 645-650
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Tidskriftsartikel (refereegranskat)abstract
- (A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees. The UvrB-DNA complex is then recognized by UvrC) and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy. Both methods reveal that the mutant UvrB is unable to bend DNA.
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- Johansson, Tomas, et al.
(författare)
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X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli.
- 2005
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 352:2, s. 299-312
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Tidskriftsartikel (refereegranskat)abstract
- The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
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