Ga3+ as a mechanistic probe in Fe3+ transport: Characterization of Ga3+ interaction with FbpA

Journal Article

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 Ga 3+ and Fe3+, focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga 3+ 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′eff = 13.7 ± 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 Fe 3+ into apo- or Ga3+-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga3+ transport and toxicity in cell culture studies. © 2008 SBIC.

Full Text

Duke Authors

Cited Authors

  • Weaver, KD; Heymann, JJ; Mehta, A; Roulhac, PL; Anderson, DS; Nowalk, AJ; Adhikari, P; Mietzner, TA; Fitzgerald, MC; Crumbliss, AL

Published Date

  • 2008

Published In

Volume / Issue

  • 13 / 6

Start / End Page

  • 887 - 898

International Standard Serial Number (ISSN)

  • 0949-8257

Digital Object Identifier (DOI)

  • 10.1007/s00775-008-0376-5