title: Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-β-globin subunit complex.
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 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ]222) and difference absorption coefficient at 406nm (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]222and Δε406) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ]222and Δε406were 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.
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
authors: Wahiduzzaman
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
authors: Mohammad Aasif
authors: MA
authors: Dar
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
authors: Mohd
authors: M
authors: Amir
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
authors: Asimul
authors: A
authors: Islam
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India.
authors: Md Imtaiyaz
authors: MI
authors: Hassan
authors: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India. Electronic address: fahmad@jmi.ac.in.
authors: Faizan
authors: F
authors: Ahmad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D019791:Guanidine / Q000737:chemistry; D007668:Kidney / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification; D012756:Sheep; D013050:Spectrometry, Fluorescence; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000302:isolation & purification; D055544:beta-Globins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D010446:Peptide Fragments; D021122:Protein Subunits; D055544:beta-Globins; D014305:Triose-Phosphate Isomerase; D019791:Guanidine
keywords: Column chromatography; GdmCl-induced denaturation; Globin β-chain; Protein purification; Protein stability; Triose phosphate isomerase