Metal-Binding Properties of Human Centrin-2 Determined by Micro-Electrospray Ionization Mass Spectrometry and UV Spectroscopy

Theodore A. Craig, Linda M. Benson, H. Robert Bergen, Sergei Y. Venyaminov, Jeffrey L. Salisbury, Zachary C. Ryan, James R. Thompson, Justin Sperry, Michael L. Gross, Rajiv Kumar

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (μESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Δ25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.

Original languageEnglish (US)
Pages (from-to)1158-1171
Number of pages14
JournalJournal of the American Society for Mass Spectrometry
Volume17
Issue number8
DOIs
StatePublished - Aug 2006

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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