NMR measurements were performed using AVANCE 800 and AVANCE 500 spectrometers each equipped with a 5-mm triple-resonance cryogenic probe (Bruker BioSpin, Fallanden, Switzerland). The NMR data were processed and analyzed using TopSpin (Bruker BioSpin) and SPARKY software26 .
The chemical shifts of the FVIII fragment (Asn776–Asp838) were assigned by a standard protocol18 (link),25 (link). To obtain the dissociation constant based on NMR peak attenuation, 0.1 mM [15N]FVIII fragment was titrated with 0.02–0.3 mM of MCFD2/ERGIC-53CRD or MCFD2 only in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.0) containing 10 mM CaCl2, 150 mM NaCl, and 10% (v/v) D2O. Regarding [15N]MCFD2, NMR experiments were performed as described previously18 (link); two molar excess amounts of peptides corresponding to Asp926–Lys953 of FV and Asn776–Asp838 of FVIII were individually added to 0.2 mM [15N]MCFD2. These experiments were performed in the presence or absence of five molar excess amounts of a tridecapeptide derived from Arg44–His56 of ERGIC-53 or that from Arg31–Arg44 of ERGL.
The chemical shifts of the FVIII fragment (Asn776–Asp838) were assigned by a standard protocol18 (link),25 (link). To obtain the dissociation constant based on NMR peak attenuation, 0.1 mM [15N]FVIII fragment was titrated with 0.02–0.3 mM of MCFD2/ERGIC-53CRD or MCFD2 only in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.0) containing 10 mM CaCl2, 150 mM NaCl, and 10% (v/v) D2O. Regarding [15N]MCFD2, NMR experiments were performed as described previously18 (link); two molar excess amounts of peptides corresponding to Asp926–Lys953 of FV and Asn776–Asp838 of FVIII were individually added to 0.2 mM [15N]MCFD2. These experiments were performed in the presence or absence of five molar excess amounts of a tridecapeptide derived from Arg44–His56 of ERGIC-53 or that from Arg31–Arg44 of ERGL.
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