The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Spring 3-31-2019 NaAtm1: Examining the mechanism of a Heavy- metal ABC Exporter Dennis Hicks University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biochemistry Commons Recommended Citation Hicks, Dennis, "NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter" (2019). Master's Theses. 1204. https://repository.usfca.edu/thes/1204 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter A thesis presented to the faculty of the Department of Chemistry at the University of San Francisco in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Written by Dennis Hicks Bachelor of Science in Biochemistry San Francisco State University August 2019 NaAtm1: Examining the mechanism of a Heavy-metal ABC Exporter Thesis written by Dennis Hicks This thesis is written under the guidance of the Faculty Advisory Committee, and approved by all its members, has been accepted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at the University of San Francisco Thesis Committee Janet G. Yang, Ph.D. Research Advisor Ryan M. West, Ph.D. Assistant Professor Nicole M. Thometz, Ph.D. Assistant Professor Marcelo F. Camperi, Ph.D. Dean, College of Arts and Sciences Acknowledgements They say it takes a village to raise a child and this can’t be more true for this thesis and my time at USF. I would like to sincerely thank my advisor Dr. Janet Yang for her patience, support, and guidance (along with all of the baby things for Rosie). Her immense knowledge and ability to easily communicate complex ideas has made my education at USF very enjoyable. I would like to give a special thanks to Jeff Oda, whose incredible knowledge of where stuff is in the chemistry department has saved the Yang lab countless hours and an untold amount of money. This thesis wouldn’t have included as much as it does without the help of all current and former members of the Yang Lab and for that I am eternally grateful. Their help and company made life in the Yang lab amazing. I would also like to thank my wife Kylie, for her undying support and eternal patience. She has no idea how grateful I am for her support and help during my time at USF, as well as taking the nightshift with our daughter Rosie. Table of Contents 1 Introduction ..............................................................................................................................1 1.1 A Brief history of ABC transporters ........................................................................1 1.2 ABC Transporters in human health .........................................................................4 1.2.1 Multi-drug resistant (MDR) abilities of cancer and bacteria/fungi .........................4 1.2.2 Human ABC transporter related diseases ................................................................5 1.3 Challenges in organizing the ABC transporter family .............................................7 1.4 Basic structure and components of ABC transporters .............................................7 1.4.1 The transmembrane domains (TMDs) .....................................................................9 1.4.2 The nucleotide binding domains (NBDs) ................................................................9 1.4.2.1 Walker A motif (P-loop) ..................................................................................12 1.4.2.2 Walker B motif ................................................................................................13 1.4.2.3 A-loop ..............................................................................................................14 1.4.2.4 C Motif (ABC signature motif)........................................................................15 1.4.2.5 D-loop ..............................................................................................................16 1.4.2.6 H-loop ..............................................................................................................17 1.4.2.7 Q-loop ..............................................................................................................18 1.5 Accessory Domains ..........................................................................................................19 1.5.1 Extracytoplasmic domains .....................................................................................19 1.5.2 Membrane-embedded domains ..............................................................................20 1.5.3 Cytosolic regulatory domains ................................................................................20 1.5.4 Accessory catalytic domains ..................................................................................21 1.6 ABC Transporter classes........................................................................................21 1.7 ABC importer classes and proposed mechanisms .................................................22 1.7.1 Structure of Type I and II importers ......................................................................23 1.7.2 Mechanisms of Type I and II importers .................................................................25 1.7.3 Structure of Type III (ECF) importers ...................................................................29 1.7.3.1 Differences between ECF subgroups I and II ..................................................30 1.7.3.2 Type III substrates............................................................................................30 1.7.4 Mechanism of Type III importers ..........................................................................31 1.8 ABC exporter classes and proposed mechanism ...................................................32 iv 1.8.1 ABCA ....................................................................................................................33 1.8.2 ABCB .....................................................................................................................33 1.8.3 ABCC .....................................................................................................................33 1.8.4 ABCD ....................................................................................................................34 1.8.5 ABCE .....................................................................................................................34 1.8.6 ABCF .....................................................................................................................34 1.8.7 ABCG ....................................................................................................................35 1.8.8 Proposed exporter mechanism ...............................................................................36 1.9 ABC non-porters ....................................................................................................37 1.10 Protein of interest: an ABC exporter found in mitochondria .................................37 1.10.1 Atm1 in Novosphingobium aromaticivorans .........................................................39 1.10.2 NaAtm1 substrate binding sites .............................................................................41 1.10.3 In vitro characterization of NaAtm1 activity .........................................................42 1.10.4 In vivo functionality of NaAtm1 ............................................................................43 1.11 Studies presented in this work ...............................................................................43 1.12 References ..............................................................................................................44 2 Protein Purification ...............................................................................................................54 2.1 Method theories .....................................................................................................54 2.1.1 Immobilized metal affinity chromatography (IMAC) ...........................................55 2.1.1.1 Poly-histidine tags ............................................................................................56 2.1.2 Size exclusion chromatography (SEC) ..................................................................57 2.1.3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ...............59 2.2 Specialized instruments .........................................................................................62 2.2.1 Fast protein liquid chromatography (FPLC) instrument ........................................63 2.2.2 Spectrophotometer .................................................................................................64 2.3 Purification of NaAtm1 ..........................................................................................65 2.3.1 NaAtm1 purification overview ..............................................................................65