The N -terminal sequence of Escherichia coli CL synthase: Function(s) of the conserved residues.
Item
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Title
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The N -terminal sequence of Escherichia coli CL synthase: Function(s) of the conserved residues.
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Identifier
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AAI3283204
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identifier
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3283204
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Creator
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Quigley, Bernard R.
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Contributor
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Adviser: Burton E. Tropp
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Date
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2007
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Escherichia coli (E. coli) cardiolipin synthase (CL synthase) was fused to a C-terminal 6 X histidine tag. The fusion protein migrates at the predicted apparent molecular mass when analyzed by SDS PAGE and western blot, and has little to no in vivo or in vitro activity. Removal of the tag by reintroduction of the C-terminal stop codon restores full activity to CL synthase in vivo..;An EE (EYMPME) epitope tag was introduced into the primary sequence of CL synthase by making three relatively conservative mutations. The EE epitope tagged enzyme migrates at the same apparent molecular mass as the wild type CL synthase when analyzed by SDS PAGE, and is recognized on western blot using an anti EE antibody, whereas the wild type is not. The EE tagged and wild type proteins have nearly identical activity in vivo and similar activity in vitro..;Mutations were made to amino acid residues within the N-terminus of the EE epitope tagged CL synthase, and their effect on the processing of CL synthase was analyzed by SDS PAGE and western blot. The in vivo and in vitro activity of CL synthase with these mutations was also analyzed. Mutations made within the N-terminus of CL synthase resulted in five classes of mutants: (i) those with an increase in apparent molecular mass that have activity in vitro but not in vivo, (ii) those with an increase in apparent molecular mass that have no activity in vivo or in vitro, (iii) those with an increase in apparent molecular mass that have activity in vivo and in vitro, (iv) those with a decrease in apparent molecular mass that have no activity in vivo or in vitro, and (v) those mutations that have little to no effect on the processing or activity of CL synthase. A single mutant (pBQ72, LV7-8SS) was found that has in vitro, but not in vivo activity, suggesting an alteration in membrane topology. Furthermore, this mutant CL Synthase (LV7-8SS) migrates with an increase in apparent molecular mass relative to the EE tagged CL synthase with a wild type N-terminus, indicative of an alteration in processing.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
