Abstract
Zinc modulates the biological function of histidine-rich glycoprotein (HRG) through binding to its His-rich region (HRR). The Zn2+-binding properties of a 35 amino-acid biologically-active peptide mimic of the HRR, HRGP330, were investigated using dissociative mass spectrometry approaches in addition to travelling-wave ion mobility mass spectrometry (TWIM-MS). Native mass spectrometry confirmed zinc binding to HRGP330; however, broadening of the 1H NMR resonances upon addition of Zn2+ ions precluded the attainment of structural information. A complementary approach employing TWIM-MS indicated that HRGP330 has a more compact structure in the presence of Zn2+ ions. Top-down MS/MS data supported a metal-binding-induced conformational change, as fewer fragments were observed for Zn2+-bound HRGP330. Zn2+-bound fragments of both N-terminal and C-terminal ends of the peptide were identified from collision-induced dissociation (CID) and electron transfer dissociation/proton transfer reaction (ETD/PTR) experiments, suggesting that multiple binding sites exist within this region of HRG. The combination of mass spectrometry and NMR approaches provides new insight into the highly dynamic interaction between zinc and this His-rich peptide.