Biochemical and Biophysical Characterization of Bacterial Transition Metal Transporters by Functional Reconstitution in Artificial Lipid Bilayers

Abstract

Transition metals play a vital role in all living organisms due to their key structural and functional properties central to diverse metabolic processes. However, because of their high reactivity organisms have evolved sophisticated biomolecular protein networks to control intercellular metal ion homeostasis, without reaching toxic intracellular levels. Transmembrane transporter proteins play a gate-keeper role in maintaining the dynamic flux of these transition metal ions across biological membranes, thereby finely tuning metal delivery and availability in cells and subcellular organelles. P-type ATPases are a superfamily of transmembrane primary active transporters involved in translocating substrates against an electrochemical gradient which play a key role in maintaining homeostasis of cellular concentrations of essential ions. They are classified into 5 classes (P1, P2, P3, P4 and P5) based on their substrate selectivity. This dissertation is focused on studying the substrate selectivity and mechanism of translocation in the P1B class of P-type ATPases which are involved in transition metal transport for both essential and toxic transition metals. P1B-type ATPases are classified into 7 sub families (P1B-1 - P1B-7 types) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub-family’s substrate selectivity, resulting in the existence of pumps that can selectively translocate 1st, 2nd and 3rd row transition metals across the lipid bilayer. Considering their central role in controlling cellular metal levels and extrusion in cells they are also acting as virulence factors in pathogenic bacteria. Studies towards their characterization could therefore help in establishing them as new potential therapeutic targets to develop novel antibiotics to overcome the bacterial resistance observed with traditional antibiotics. However, the substrate transport across lipid bilayers, the overall mechanism for cargo translocation, and kinetics of these transporters remain elusive to a significant extent, due to lack of molecular tools to study putative metal substrate transport across membranes in real-time in a native-like environment. In light of this, metal-stimulated ATPase activity assays were coupled with an experimental platform based on multiple fluorescence sensor probes, to study substrate selectivity, transport mechanism, including counterion transport and electrogenicity, and translocation kinetics in realtime with recombinantly expressed proteins belonging to P1B-1 (CopA from E. coli) and P1B-5 (Nia from S. meliloti) classes reconstituted in artificial lipid bilayer vesicles known as proteoliposomes. The proteoliposomes were used as an in-vitro tool to determine metal selectivity and the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn-on florescence signal upon substrate ion binding and translocation. However, the use of the proteoliposomes is challenging due to their intrinsic structural instability and susceptibility to stressors like temperature, aging, and chemicals, which limits their shelf life. Therefore, an experimental approach was developed to stabilize membrane proteins and proteoliposomes by encapsulating them in a sheddable metal organic framework, which reduces their susceptibility to external stressors. This platform sheds light on developing methods to utilize proteoliposomes in biochemical and biophysical investigation of transmembrane proteins and in drug delivery applications. In addition, this approach would help to overcome the challenges of cold-chain therapeutic transport of liposomal vaccine formulations.