title: Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.
abstract: Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
authors.forename: J R
authors.initials: JR
authors.lastname: Wu
authors.forename: B R
authors.initials: BR
authors.lastname: Lentz
mesh_terms: D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology*; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D005583:Fourier Analysis; D007700:Kinetics; D008081:Liposomes; D008961:Models, Structural; D010446:Peptide Fragments / Q000737:chemistry* / Q000187:drug effects; D010743:Phospholipids / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry* / Q000187:drug effects; D011516:Prothrombin / Q000737:chemistry* / Q000187:drug effects; D013055:Spectrophotometry, Infrared / Q000379:methods
chemical_list: D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D011498:Protein Precursors; C029733:prothrombin fragment 1; D011516:Prothrombin; D004492:Edetic Acid; D002122:Calcium Chloride