Investigating Native Metal Ion Binding Sites in Mammalian Histidine-Rich Glycoprotein

16 March 2023, Version 3
This content is a preprint and has not undergone peer review at the time of posting.


Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterisation. Binding of divalent metal ions, particularly zinc(II), to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterisation of the zinc(II) binding sites of HRG is a challenge; their number and composition, as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To our best knowledge this is the first time this suite of EPR techniques has been applied to count and characterise endogenous metal ion binding sites in a native mammalian protein of unknown structure.


EPR spectroscopy
hyperfine spectroscopy
metal ion binding proteins
plasma proteins

Supplementary materials

Supplementary Information
Detailed experimental procedures, HRG secondary structure prediction, CW EPR data, hyperfine spectroscopy and PDS data, and the derivation of the speciation model.


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