Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy.
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 (muESI-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. Delta 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 structure.
Craig
Theodore A
TA
Department of Medicine, Mayo Clinic College of Medicine and Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Benson
Linda M
LM
Bergen
H Robert
HR
Venyaminov
Sergei Y
SY
Salisbury
Jeffrey L
JL
Ryan
Zachary C
ZC
Thompson
James R
JR
Sperry
Justin
J
Gross
Michael L
ML
Kumar
Rajiv
R
D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D018797:Cell Cycle Proteins / Q000737:chemistry*; D003198:Computer Simulation; D008670:Metals / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D008670:Metals