Table 1.
Fig 1.
AF4 fractograms and molecular weight (MW) of (a) HSA, (b), GA-Z and (c) Berry plot of GA-Z in 25 mM phosphate buffer at pH 7.
Fig 2.
(a) AF4-UV fractograms and hydrodynamic radius (dH) of the complex between GA-Z and HSA with different mixed mole ratios The gray and black dashed lines are fractograms of pure GA-Z and HSA, and the solid lines are fractograms of each mixed mole ratio (red = 0.03, orange = 0.14, dark yellow = 0.28, green = 0.85, blue = 1.42, purple = 2.83, and pink = 5.66). (b) The calculated binding mole ratio from each UV detector response GA-Z bound to total amount of HSA (square), GA-Z bound to monomeric albumin (circle) GA-Z bound to dimeric HSA (diamonds).
Fig 3.
Experimental SAXS data of GA-Z at three different concentrations 9 mg/ml (circles), 6 mg/ml (triangles), 3 mg/ml (squares).
Table 2.
Fig 4.
Size-exclusion chromatography chromatogram of GA-Z (dashed magenta), HSA (dotted black), GA-Z-HSA (line red).
Fig 5.
SEC-SAXS data and modeling of the GA-Z dimer.
(a) Experimental data is shown as white circles with the fits using Coral (red line) and EOM (dashed blue line) modeling. (b) Coral rigid body model results corresponding to the fit in (a), superimposed on the ab initio model using DAMMIF on the same experimental data. The model is shown also after a 90 ° rotation. (c) Results from the EOM modeling, shown in grey filling is the random pool from which models could be chosen to fit the data in (a). The black line corresponds to the frequency of models with a certain radius of gyration that was used in the final fit in (a) to the experimental data. (d) Four representative models from the EOM modeling procedure, shown in ribbon representation.
Fig 6.
(a) HSA and GA-Z-HSA experimental data is shown as white circles and white squares, respectively. The corresponding fit to the HSA experimental data using Crysol and an X-ray crystallography model with PDB ID: 1TF0 is shown with a magenta line. For fitting to the GA-Z-HSA experimental we show both the Crysol fit (magenta line) using only HSA PDB ID: 1TF0 as well as that from the Coral rigid body model (red line), for comparison. (b) The resulting rigid body model from Coral is shown in a ribbon representation with HSA in green and GA-Z in blue, the linker between the GA and Z-domains is shown in magenta. GA-Z is bound to the HSA DII domain.