Calcium Binding to a Factor IX Fc Fusion Protein and Effects on Higher‐Order Structure

Conformational Comparability of Factor IX–Fc Fusion Protein, Factor IX, and Purified Fc Fragment in the Absence and Presence of Calcium

“…Thus, our H/DX‐MS data show that the conformation of the Fc fragment alone is not influenced by the presence of Ca 2+ ions. This result supports our above DSC results as well as earlier studies by Li et al23 involving the analysis of Ca 2+ binding to rFIX–Fc and Fc fragment using ITC.…”

“…Structural characterization of FIX domains and its calcium binding properties have been extensively studied 11,21,23,28. However, a detailed characterization and understanding of the structure and structural dynamics of an intact rFIX or rFIX–Fc and the effect of binding Ca 2+ on these proteins are limited.…”

“…For activation of FIX and the FIX part of rFIX–Fc, both require the binding of calcium (Ca 2+ ) ions, which interacts (with differential affinities) to the Gla, EGF, and serine protease domains. 20–24 For rFIX, an array of different methods has been used to probe the structure of various rFIX domains and changes to the structure of these domains as a result of Ca 2+ binding. These methods include amino acid substitution,25 X‐ray crystallography,26 nuclear magnetic resonance,27 spectroscopy, and calorimetry 23,28.…”

“…20–24 For rFIX, an array of different methods has been used to probe the structure of various rFIX domains and changes to the structure of these domains as a result of Ca 2+ binding. These methods include amino acid substitution,25 X‐ray crystallography,26 nuclear magnetic resonance,27 spectroscopy, and calorimetry 23,28. However, to our knowledge, detailed characterization of the structure and structural dynamics of an intact rFIX–Fc fusion protein, or for that matter rFIX, in solution with or without Ca 2+ is limited.…”

“…However, to our knowledge, detailed characterization of the structure and structural dynamics of an intact rFIX–Fc fusion protein, or for that matter rFIX, in solution with or without Ca 2+ is limited. In addition, the structural comparison between rFIX and the FIX region of the fusion protein rFIX–Fc is scarce,23 as is the comparison between Fc and the Fc region of rFIX–Fc.…”