Structure of the propeptide of prothrombin containing the .gamma.-carboxylation recognition site determined by two-dimensional NMR spectroscopy

• Published in: Biochemistry (Easton) 1991-10, Vol.30 (41), p.9835-9841
• Main Authors: Sanford, David G, Kanagy, Carolyn, Sudmeier, James L, Furie, Barbara C, Furie, Bruce, Bachovchin, William W
• Format: Article
• Language: English
• Subjects: 550600 - Medicine; Amino Acid Sequence; AROMATICS; BARYONS; Binding Sites; Biological and medical sciences; BLOOD COAGULATION FACTORS; Blood coagulation. Blood cells; Carbon-Carbon Ligases; CARBOXYLATION; CHEMICAL REACTIONS; Circular Dichroism; COAGULANTS; Coagulation factors; Cold Temperature; DICHROISM; DRUGS; ELEMENTARY PARTICLES; Enzyme Stability - drug effects; FERMIONS; Fundamental and applied biological sciences. Psychology; gamma -carboxylation; HADRONS; HEAVY WATER; HEMATOLOGIC AGENTS; HEPARIN ANTAGONISTS; HYDROGEN COMPOUNDS; Ligases - chemistry; MAGNETIC RESONANCE; Magnetic Resonance Spectroscopy; Molecular and cellular biology; Molecular Sequence Data; MOLECULAR STRUCTURE; NUCLEAR MAGNETIC RESONANCE; NUCLEONS; ORGANIC COMPOUNDS; ORGANIC OXYGEN COMPOUNDS; OXYGEN COMPOUNDS; Peptide Fragments - chemical synthesis; PEPTIDES; Protein Conformation; Protein Precursors - chemistry; PROTEINS; PROTHROMBIN; Prothrombin - chemistry; PROTONS; QUINONES; RADIOLOGY AND NUCLEAR MEDICINE; RESONANCE; Trifluoroethanol - pharmacology; VITAMIN K; VITAMINS; WATER
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00105a004

The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.