On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy.
The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.
Gettins
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Wooten
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EW
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation
D006146:Guanidines; D000990:Antithrombin III; D006493:Heparin; D019791:Guanidine