Functional Reconstitution and in-vitro Characterization of Bacterial Transmembrane Transition Metal Transporters

Transition metals play a pivotal role in all kingdoms of life since they are involved in crucial biochemical reactions as enzyme co-factors, or they perform structural roles in biomolecules. However, due to their reactivity, high levels of transition metals can be toxic to cells. Transmembrane transporter proteins play a “gate-keeper” role in maintaining cellular metal homeostasis. However, the molecular mechanisms of many transporters remain elusive due to lack of biochemical tools to study them at molecular level. This dissertation discusses the development of biochemical and biophysical tools to study membrane transporters to reveal their transport mechanisms, metal-substrate recognition, and energetics. Iron plays a central role in life due to its critical role as an enzyme cofactor in metabolic processes. IroT/MavN is a putative iron transporter and a virulence factor at the host-pathogen interface in Legionella pneumophilla, which allows intravacuolar iron acquisition from the host cell, allowing iron mobilization and preventing insolubility in its ferric oxidation state. The substrate selectivity and mechanism of transport of IroT/MavN was revealed by incorporating recombinantly purified protein in artificial lipid bilayer vesicles, (proteoliposomes) which mimic the native lipid bilayer environment. The high-affinity ferrous iron transport mechanism of IroT/MavN which selectively transports Fe2+ over other transition metals, was revealed by encapsulating the metal turn-on fluorescent probe Fluozin-3 in proteoliposome lumen. Mutational analysis revealed the critical amino acid residues for metal recognition, binding, and translocation. Moreover, the proton coupled counter-transport mechanism of IroT/MavN was revealed by using the pH probe pyranine and the Fe2+ -H + antiporter mechanism of IroT/MavN was demonstrated. P1B-type ATPases are primary active metal pumps, involved in maintaining metal homeostasis. Based on the metal-stimulated ATP assays, the Zn2+ pumps, (P1B-2-type) exhibit promiscuity for divalent metal ions of Zn2+/Cd2+/Hg2+/Pb2+ due to coordination and structural plasticity in their transmembrane metal binding site. Yet, the direct translocation of these ions and the transport mechanism remain largely elusive. The proteoliposome based platform was extended to study substrate promiscuity and transport mechanism in the Zn2+ pump of Cupriavidus metallidurans (CmZntA). By encapsulating a diverse set of fluorescent probes responsive to different stimuli in proteoliposome lumen, the ATP-dependent translocation for Pb2+, Zn2+, Cd2+ and Hg2+ were demonstrated and correlate to the ATP hydrolysis rates, and substrate translocation is electrogenic and not coupled to proton counter-transport. Structural and functional properties of proteoliposomes are detrimentally affected by temperature, aging, and chemical stressors due to their metastable nature and intrinsic instability of membrane proteins, limiting their utilization. To overcome these stability issues, together with collaborators, a methodology to stabilize membrane proteins and proteoliposomes was developed via a biomineralization-like process through formation of crystalline metal-organic framework (MOF) exoskeletons. Structural and functional properties were minimally affected when subjected to temperature, aging and denaturant stressors when protein-micelles and proteoliposomes were encapsulated in zeolitic imidazole framework-8 (ZIF-8) scaffolds, with a release strategy by treatment of EDTA. This platform opens new avenues in the use of liposomes for drug delivery and vaccine applications and can be utilized to overcome the challenges of “cold-chain” therapeutic transport.