Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

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 (ESI-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. ⌬25 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

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

“…Protein concentration was determined conventionally by absorbance at 277 nm using an E m M ϭ 1.46 mM Ϫ1 cm Ϫ1 and after purification by the Bio-Rad (Bradford) method (28). Native HsCen-2-HsCen-2 was expressed and purified as described earlier (29). Crystallization trials were conducted using hanging drop vapor diffusion methods with over 3000 crystallization solutions, which were then optimized for the production of large single crystals.…”

“…Titrating HsCen-2 (26,43) and other centrins (44) to Ca 2ϩ saturation is complicated by aggregation of high order oligomers, an aggregation of reversible nature. Deletion of amino acids 1-24 has little effect on protein stability, global secondary structure, or metal binding (29) but does prevent aggregation; thus, these residues must be involved in the self-assembly (45). The formation of fibers consisting of centrin may be of physiological importance in the duplication and segregation of microtubule organizing centers (21).…”

The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two‐state transition

“…Electrospray ionization mass spectrometry (ESI-MS) is a well established method for characterizing protein structural transition (Hooke et al 1995;Konermann 2004;Kaltashov and Eyles 2005;Craig et al 2006). The technique has also been applied in studying the amyloid fibrillation of amyloidogenic proteins, lysozyme in particular (Nettleton and Robinson, 2000;Caddy and Robinson 2006).…”

Architecture of the human XPC DNA repair and stem cell coactivator complex

“…Structure prediction analysis (14), limited proteolysis (14), and NMR (37) identified several highly disordered regions: the N terminus (residues 1-154), the C terminus (residues 816-940), and a loop inserted into the TGD domain comprising residues 331-517. Finally, CETN2 may also contribute to this overall flexibility, because it can adopt different conformations depending on its metal-binding state (38); however, the resolution of the XPC-RAD23B subcomplex was not markedly improved, suggesting that this contribution to complex flexibility is minor, as would be expected for its relatively small mass contribution to the complex. The recently described requirement of RNA for the XPC complex to interact with its transcription partner SOX2 (25) invokes the idea of low-complexity domains or regions, perhaps interspersed throughout XPC, linking their inherent flexibility to a critical aspect of the XPC complex's function.…”