Determination of the solution structure of the N-terminal EGF-like domain of human blood clotting factor IX and investigation of its calcium binding site using two-dimensional-NMR techniques.
Item
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Title
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Determination of the solution structure of the N-terminal EGF-like domain of human blood clotting factor IX and investigation of its calcium binding site using two-dimensional-NMR techniques.
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Identifier
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AAI9908317
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identifier
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9908317
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Creator
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Gong, Yumin.
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Contributor
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Adviser: William V. Sweeney
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Date
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1998
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Analytical | Chemistry, Physical | Biology, Molecular
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Abstract
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2D-NMR techniques have been used to determine the solution structure of a 43-amino acid synthetic peptide, that is homologous to the portion (45-87) of the N-terminal EGF-like domain of human blood coagulation factor IX (FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}). The solution structures of FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}, FX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}, mEGF and hTGF{dollar}\alpha{dollar} are compared. The results show that their tertiary structures are strikingly similar, with the exception of the termini. A previously reported structure of factor IX (46-84) does not conclude the second beta-sheet in our structure due to a shorter sequence.;Among FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}, FX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}, mEGF and hTGF{dollar}\alpha{dollar}, FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar} and FX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar} have the highest sequential similarity, while FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar} and mEGF have the highest structural similarity. The six cysteines and a few consensus residues should be important for the structural integrity of these EGF-like structures. A few residues conserved only in EGF and TGF{dollar}\alpha{dollar} may be functionally distinct. It is likely that the different roles of these EGF-like polypeptides in biological function is due to the differences of the electronic environment of the surface.;The chemical shift titration was used to identify possible calcium ligands. The results show that Asp3, Gln6 and Gly4 are likely to be involved in calcium binding. The possibility of the sidechain of Asp20 for calcium ligand is ambiguous due to the complicated titration curve. The pH titration data is insufficient to determine if the backbone carbonyls of Asp5, and Asp20 are calcium ligands. A potential calcium binding site between the sidechains of Asp3, Gln6, Asp20, and backbone of Gly4 was inferred from a visual inspection of the solution structure of FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}. The backbone carbonyls of Asp5 and Asp20 are pointed away from the potential calcium binding site, making them less likely to be calcium ligands. The potential calcium binding site of FIX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar} from this study is similar to that suggested by a previous crystal structure study. This potential calcium binding site is similar to that proposed for FX-EGF{dollar}\rm\sb{lcub}N{rcub}{dollar}. The involvement of Asp3, Gln6, and Asp20 in calcium binding is consistent with the finding that mutations occur at these specific residue positions in hemophilia B patients.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
