Substitution of Glutamate Residue by Lysine in the Dimerization Domain Affects DNA Binding Ability of HapR by Inducing Structural Deformity in the DNA Binding Domain
Figure 1
Analytical molecular sieve chromatography to determine the molecular weight of the functional (HapRV2G) and non-functional proteins (HapRV2 and HapRV2G-E117K).
(A) A calibration curve of column was obtained by plotting the Kav of the protein standards against the Mr of the standards. Six GE-Healthcare gel filtration molecular mass markers were used: Blue dextran (200 kDa), Conalbumin (75 kDa), Ovalbumin (44 kDa), Carbonic anhydrase (29 kDa), Ribonulcease A (13.7 kDa) and Aprotinin (6.5 kDa). Each marker along with its Mr is represented by a unique colour. Kav values for all the proteins obtained from the Eq. 1 were fitted to calibration curve to estimate the molecular weight of proteins (∼52 kDa). (B) The elution profiles of the functional (HapRV2G) and non-functional proteins (HapRV2 and HapRV2G-E117K). Column was equilibrated in 10 mM Tris, pH7.9, 100 mM KCl, and 0.1 mM EDTA. Elution profile of Carbonic anhydrase (29 kDa) is shown in dotted line for comparison.