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- Title
Ga<sup>3+</sup> as a mechanistic probe in Fe<sup>3+</sup> transport: characterization of Ga<sup>3+</sup> interaction with FbpA.
- Authors
Weaver, Katherine D.; Heymann, Jared J.; Mehta, Arnav; Roulhac, Petra L.; Anderson, Damon S.; Nowalk, Andrew J.; Adhikari, Pratima; Mietzner, Timothy A.; Fitzgerald, Michael C.; Crumbliss, Alvin L.
- Abstract
The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe3+ from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga3+, which, unlike Fe3+, is not redox-active. Because of a similar size and charge as Fe3+, Ga3+ is widely used as a non-redox-active Fe3+ substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga3+ and Fe3+, focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga3+ complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH2PO4 was determined by UV-difference spectroscopy as $$ \log \,K_{{\text{eff}}}^\prime = 13.7 \pm 0.6. $$ This represents a 105-fold weaker binding relative to Fe3+ at identical conditions. The unfolding/refolding behavior of Ga3+ and Fe3+ holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe3+ and Ga3+ sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga3+FbpA–PO4 assembly, and the similarities/differences of stepwise loading of Fe3+ into apo- or Ga3+-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga3+ transport and toxicity in cell culture studies.
- Subjects
ATP-binding cassette transporters; CARRIER proteins; ENZYME kinetics; PROTEIN folding; PATHOGENIC microorganisms; QUALITATIVE chemical analysis
- Publication
Journal of Biological Inorganic Chemistry (JBIC), 2008, Vol 13, Issue 6, p887
- ISSN
0949-8257
- Publication type
Article
- DOI
10.1007/s00775-008-0376-5