Genetic manipulation of P-glycoprotein provides new tools for biophysical studies of the ABC multidrug transport mechanism

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Genetic manipulation of P-glycoprotein provides new tools for biophysical studies of the ABC multidrug transport mechanism

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Title: Genetic manipulation of P-glycoprotein provides new tools for biophysical studies of the ABC multidrug transport mechanism
Author: Swartz, Douglas
Abstract: P-glycoprotein (Pgp) is an ATP Binding Cassette (ABC) transporter that functions as a multidrug efflux pump, and contributes to multidrug resistance in cancer and other diseases. Pgp operates through a poorly understood ATP dependent polyspecific transport mechanism that allows it to bind and transport a wide variety of structurally unrelated drugs. Understanding Pgp drug binding and transport would allow rational design of novel Pgp inhibitors and provide a better understanding of the general ABC transport mechanism. In this work, Pgp was genetically modified to build two new tools for biophysical studies: a tryptophan (Trp)-less Pgp that can be used for site-specific Trp fluorescence studies of Pgp drug binding sites, and a cysteine (Cys)-less Pgp that can be labeled with spectroscopic probes for measuring the intramolecular distances needed to identify distinct Pgp protein conformations. Initial efforts to replace the eleven endogenous Pgp Trps with another aromatic amino acid demonstrated that multiple Trps could be removed from Pgp while maintaining protein function, but also suggested that aromatic residues are not always the best Trp replacements. Therefore, a directed evolution procedure was developed to determine which amino acids could replace each endogenous Pgp Trp. Site-saturation mutagenesis simultaneously replaced blocks of 3 or 4 Pgp Trps with the 19 other amino acids. The mutants were subjected to a stringent selection in yeast to determine which amino acids could replace each Trp. These mutants were then combined into full-length Pgp Trp mutants and re-selected. This approach successfully identified several Trp-less and minimal Trp Pgp mutants, which contain one or two native Trps in positions suitable for drug binding studies. Similarly, directed evolution was used to remove Cys residues from a codon optimized Pgp gene that was previously designed and characterized. Directed evolution revealed that the preferred amino acid substitutions were location specific and generally biased towards non-conserved amino acids such as glycine and proline. While this work successfully produced two new tools for studying Pgp, it also demonstrates that removing conserved amino acids, such as Trp and Cys, from a protein is highly dependent on the local environment of the residue being replaced.
URI: http://hdl.handle.net/2346/47087
Date: 2012-12

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