Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein
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Case Western Reserve University School of Graduate Studies / OhioLINK
1993
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=case1057080659 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-case10570806592021-08-03T05:31:04Z Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein Kehres, David George Mutational kinetic analysis Escherichia coli L-arabinose binding protein The surface of the Escherichia coli L-arabinose-binding protein, ABP, that interacts with the AraG:AraH complex, the cytoplasmic membrane component of the L-arabinose periplasmic transport system, has been defined by saturation and site-specific mutagenesis. A set of ABP mutants with single or multiple amino acid substitutions has been generated. The transport phenotypes of these mutants, when expressed with wild-type AraG and AraH in whole cells, suggest that the surface containing the mouth of the arabinose-binding cleft is responsible for most if not all of ABP's interactions with AraG:AraH. Most mutations which alter transport involve replacement of one or more charged residues on this surface with neutral amino acids. In most instances charge neutralization reduces uptake, but in some cases it increases uptake. The saturating initial uptake velocity Ven and the half-saturating substrate concentration Ken vary in proportion to each other in most of these mutants. This observation has led to the development of a simple first-order kinetic model, applicable to periplasmic transport in general, and to the proposal that wild-type arabinose periplasmic transport represents a limiting case of the model in which kfor, the forward rate of transport for a docked complex between liganded ABP and AraG:AraH, greatly exceeds kund, the rate at which the liganded binding protein dissociates. Genetic dominance patterns in cells expressing two different forms of ABP indicate that the Km of translocation at the cytoplasmic membrane surface is lower for "down" mutants and higher for "up" mutants than it is for wild-type ABP, and vesicle reconstitution studies suggest that the equilibrium binding affinity of liganded wild-type ABP for AraG:AraH is between 0.025 μM and 1.0 μM, much smaller than the expected Km. Both of these results are consistent with the kinetic limit predicted by the model. Vesicle-binding studies also suggest that at least one of the transport mutants has an altered equilibrium binding affinity for AraG:AraH, which supports the contention that the ABP cleft mouth surface contacts the membrane complex directly. 1993 English text Case Western Reserve University School of Graduate Studies / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=case1057080659 http://rave.ohiolink.edu/etdc/view?acc_num=case1057080659 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Mutational kinetic analysis Escherichia coli L-arabinose binding protein |
| spellingShingle |
Mutational kinetic analysis Escherichia coli L-arabinose binding protein Kehres, David George Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| author |
Kehres, David George |
| author_facet |
Kehres, David George |
| author_sort |
Kehres, David George |
| title |
Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| title_short |
Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| title_full |
Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| title_fullStr |
Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| title_full_unstemmed |
Mutational and kinetic analysis of the Escherichia coli L-arabinose binding protein |
| title_sort |
mutational and kinetic analysis of the escherichia coli l-arabinose binding protein |
| publisher |
Case Western Reserve University School of Graduate Studies / OhioLINK |
| publishDate |
1993 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=case1057080659 |
| work_keys_str_mv |
AT kehresdavidgeorge mutationalandkineticanalysisoftheescherichiacolilarabinosebindingprotein |
| _version_ |
1719421151984222208 |