Title
Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-β-globin subunit complex
Author
Wahiduzzaman, Mohammad Aasif Dar, Mohd. Amir, Asimul Islam, Md. Imtaiyaz Hassan, Faizan Ahmad
Year
2016
Journal
International Journal of Biological Macromolecules
Abstract
Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1 Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222 nm ([θ]222) and difference absorption coefficient at 406 nm (Δε406) at pH 7.5 and 25 °C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ]222 and Δε406) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ]222 and Δε406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Chemical stability, Protein denaturation, Thermodynamics, Biochemistry