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

Published

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 Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) 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 Fe(3+), 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 KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) 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 Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

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

  • August 2008

Published In

Volume / Issue

  • 13 / 6

Start / End Page

  • 887 - 898

PubMed ID

  • 18461372

Pubmed Central ID

  • 18461372

Electronic International Standard Serial Number (EISSN)

  • 1432-1327

International Standard Serial Number (ISSN)

  • 0949-8257

Digital Object Identifier (DOI)

  • 10.1007/s00775-008-0376-5

Language

  • eng