Figure 3.
HDX-MS of FIXa irreversibly bound to the pseudo-substrate Glu-Gly-Arg-chloromethylketone (FIXaEGR). (A) FIXaEGR was compared with EGR-free FIXa where changes (mapped on FIXa crystal structure PDB: 2wpm) were detectable for hinge EGF-1/EGF-2, interface EGF-2/protease domain, 99-loopCT, 148-loopCT, 162-helixCT, and 220-loopCT. (B) FIXaEGR was incubated in the presence and absence of FVIIIa, and differences were highlighted on the FIXa crystal structure. Changes were detectable at EGF-2, interface EGF-2/protease domain, 99-loopCT, helix 126-132CT, 148-loopCT, 162-helixCT, and helix 236-240CT. HDX-MS measurements were based on 3 time points and mapped following the color coding as indicated. Differences were rated as prominent when 3 out of 3 time points showed ≥0.35 Da difference per peptide, moderate with 1 or 2 time points out of 3, and no appreciable change when all time points showed <0.35 Da difference.

HDX-MS of FIXa irreversibly bound to the pseudo-substrate Glu-Gly-Arg-chloromethylketone (FIXaEGR). (A) FIXaEGR was compared with EGR-free FIXa where changes (mapped on FIXa crystal structure PDB: 2wpm) were detectable for hinge EGF-1/EGF-2, interface EGF-2/protease domain, 99-loopCT, 148-loopCT, 162-helixCT, and 220-loopCT. (B) FIXaEGR was incubated in the presence and absence of FVIIIa, and differences were highlighted on the FIXa crystal structure. Changes were detectable at EGF-2, interface EGF-2/protease domain, 99-loopCT, helix 126-132CT, 148-loopCT, 162-helixCT, and helix 236-240CT. HDX-MS measurements were based on 3 time points and mapped following the color coding as indicated. Differences were rated as prominent when 3 out of 3 time points showed ≥0.35 Da difference per peptide, moderate with 1 or 2 time points out of 3, and no appreciable change when all time points showed <0.35 Da difference.

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