Probing protein–metal ion interactions by electrospray ionization mass spectrometry: enolase and nucleocapsid protein

✍ Scribed by Joseph A. Loo

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 401 KB
• Volume: 204
• Category: Article
• ISSN: 1387-3806
• DOI: 10.1016/s1387-3806(00)00334-1

✦ Synopsis

Electrospray ionization mass spectrometry (ESIMS) was used to study the metal ion binding stoichiometry and interactions with the proteins enolase and nucleocapsid protein (NCp7). Yeast enolase is a 93-kDa homodimeric enzyme that requires divalent metal ion binding for activity. The ESIMS studies of yeast enolase show the noncovalently bound dimer as the most abundant species for the apo and holo forms of the protein. However, the Mg 2ϩ -and Mn 2ϩ -bound enolase dimeric enzymes exhibit enhanced stability relative to their apo-and sodium-bound counterparts. Only a small proportion of the Mg 2ϩ /Mn 2ϩbound enolase dimer dissociates to the monomer state upon collisionally activated dissociation in the atmospheric pressure/vacuum interface of the ESI mass spectrometer, whereas the majority of the enolase dimer in the apo-and sodium-bound forms can be induced to dissociate to the monomer. This characteristic of the gas phase complex is consistent with its solution phase behavior. NCp7 contains two zinc finger structures. Limited proteolysis with trypsin in conjunction with ESIMS monitoring was used to determine the binding site of an initial zinc ion exposed to NCp7. The N-terminal zinc finger was found to be the primary binding site of the first zinc ion. Similar limited proteolysis experiments on the binding of NCp7-Zn 2 with the pentanucleotide d(ACGCC) suggest the participation of both zinc finger structures. This is consistent with previous solution phase binding studies using nuclear magnetic resonance and fluorescence spectroscopy. (Int J Mass Spectrom 204 (2001) 113-123) © 2001 Elsevier Science B.V.