Bioanalytical Methods Development for Detection and Characterization of Labile Metal Pools in Escherichia Coli

Low-molecular-mass (LMM) transition metal complexes are thought to play essential roles in metal ion trafficking, regulation, and signaling in biological systems. Their chemical identities remain largely unknown due to their rapid ligand-exchange rates and weak metal-ligand bonds. Lack of chemical characterization unfortunately precludes biological function. In this dissertation, methods were developed and ameliorated for an integrative approach using LC, ICP-MS, and ESI-MS to investigate the chemical nature of the labile metal pools (LMP) constituting the E. coli cytosol. An E. coli cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of metal ions from LMM metal complexes with a SEC column was minimized by pre-loading the column with 67ZnSO4. FTSs contained ca. 80 μM iron, 15 μM nickel, 13 μM zinc, 10 μM copper, and 1.4 μM manganese. FTSs chromatographically exhibited 2 – 5 iron, 2 nickel, 2 – 5 zinc, 2 – 4 copper, and 2 manganese species. Endogenous cytosolic salts suppressed ESI-MS signals, making detection of LMM metal complexes difficult. Major LMM sulfur- and phosphorus-containing species were identified, though. These included GSH, GSSG, methionine, cysteine, orthophosphate, and common mono- and dinucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the LMPs is sensitive to the nutrient metal concentration whereas treatment of the FTS with a metal chelator demonstrated the lability of these LMM metal complexes. The labile nickel pool (LNP) was selected for further identification efforts based on water-exchanging rates for transition metals. SEC-ICP-MS revealed 4 Ni-containing peaks, which were tentatively assigned to Ni(GSSG), Ni(Asp), Ni(L-His), and Ni(ATP) due to chromatographic behavior of nickel standards and results from exogenous nickel and ligand spiking into FTS. ESI-MS analysis of SEC fractions from FTS revealed Ni(GSSG), suggesting Ni(GSSG) is part of the LNP. HILIC-ESI-MS and HILIC-ICP-MS revealed Ni(GSSG) as the dominant Ni(II) species in the FTS followed by Ni(L-His)2 and then Ni(Asp)2 and Ni(ATP), showcasing the first molecular description of a LMP in E. coli.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor

Texas A&M University

Year dc:date.issued

2021

Author dc:creator

• Brawley, Hayley N

Advisor dc:contributor.advisor

• Lindahl, Paul A

Committee members dc:contributor.committeemember

• Russell, David H
• Darensbourg, Marcetta Y
• Gohil, Vishal M

• labile metal pools
• low-molecular-mass
• Inductively coupled plasma mass spectrometry
• liquid chromatography
• size-exclusion
• hydrophilic interaction
• electrospray ionization mass spectrometry
• E. coli
• nickel
• iron
• zinc
• copper
• manganese

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/1969.1/195205

OAI record oai:identifier

https://oaktrust.library.tamu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aoaktrust.library.tamu.edu%3A1969.1%2F195205