Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance

Abstract

The three-dimensional structure of recombinant desulfatohirudin in aqueous solution was determined by 1H nuclear magnetic resonance at 600 MHz and distance geometry calculations with the program DISMAN. The input for the structure calculations was prepared on the basis of complete sequence-specific resonance assignments at pH 4.5 and 22 degrees C and consisted of 425 distance constraints from nuclear Overhauser enhancements and 159 supplementary constraints from spin-spin coupling constants and from the identification of intramolecular hydrogen bonds. Residues 3-30 and 37-48 form a molecular core with two antiparallel beta-sheets and several well-defined turns. The three disulfide bonds 6-14, 16-28, and 22-39 were identified by NMR. In contrast to this well-defined molecular core, with an average root mean square distance for the polypeptide backbone of 0.8 A for a group of nine DISMAN solutions, no preferred conformation was found for the C-terminal segment 49-65, and a loop consisting of residues 31-36 is not uniquely constrained by the NMR data either. These structural properties of recombinant desulfatohirudin coincide closely with the previously described solution conformation of natural hirudin, but the presence of localized differences is indicated by chemical shift differences for residues Asp 5, Ser 9, Leu 15, Asp 53, Gly 54, and Asp 55.

Characterization, stability and refolding of recombinant hirudin

“…Although hirudin is clearly a monomer at pH 3 -4.5, as shown previously by analytical ultracentrifugation (Triebel and Walsmann, 1966) and 2D-NMR (Clore et a!., 1987;Haruyama and Wuthrich, 1989), gel filtration experiments have sugested a tendency of the inhibitor to oligomerize at neutral pH (Dodt, 1985;Konno et al, 1988;Tuong et al, 1990). The sedimentation velocity and sedimentation equilibrium studies presented above confirm the ultracentrifugation results of Triebel and Walsmann (1966) and extend them to neutral pH.…”

“…Upon determination of the complete primary structure of natural hirudin variants Harvey et al, 1986;Tripier, 1988;Scharf et al, 1989), the protein has now become available from recombinant sources in larger quantities Fortkamp et al, 1986;Harvey et al, 1986;Dodt et al, 1986;Braun et al, 1988;Crause et al, 1988;Riehl-Bellon et al, 1989) allowing a physicochemical characterization of the inhibitor purified to homogeneity. The observed ultraviolet absorption, dichroic absorption, and fluorescence emission spectra of recombinant hirudin correspond to results on its solution and crystal structure, as obtained by 2D-NMR (Folkers et al, 1989;Haruyama and Wuthrich, 1989) and X-ray crystallography (Rydel et al, 1990). The measured near-ultraviolet absorption coefficients of the inhibitor coincide with those calculated from the sequence.…”

“…Although in a number of previous studies on hirudin its molecular mass has been measured by a variety of techniques and most results were compatible with a monomeric quaternary structure (Triebel and Walsmann, 1966;Clore et al, 1987;Haruyama and Wuthrich, 1989;Bagdy et al, 1976, and references cited therein), there have been indications of a tendency of the inhibitor to associate to dimers at neutral to mildly alkaline pH (Dodt, 1985;Konno et al, 1988;Tuong et al, 1990). Thus, recombinant hirudin was subjected to sedimentation velocity and sedimentation equilibrium ultracentrifugation over a wide range of pH values ( (far-ultraviolet) hirudin at pH 3.95 to be 0.98 S (Triebel and Walsmann, 1966).…”

Conformation in solution of porcine brain natriuretic peptide determined by combined use of nuclear magnetic resonance and distance geometry

“…The problem with the introduction of hydrogen-bond information is that it is difficult to determine acceptors experimentally, hence some assumptions must be made. We adopted the evaluation method of Haruyama and Wuthrich [28] and scored the probabilities of acceptors €or every donor, as shown in Table 4. In spite of the addition of three hydrogen-bond constraints, the violation of distance constraints was reduced and structures There are two advantages of introducing energy calculations as procedures for refinement of peptide conformations.…”

Hirudin as an anticoagulant for both haematology and chemistry tests

“…Recombinant hirudin was used in our study as an anticoagulant for blood collection. Hirudins, a group of highly homologous polypeptides present in the medicinal leech (Hirudo medicinalis), have an extremely high a nity for thrombin, and are consequently potent anticoagulant s [5,6]. Recombinant hirudins have been produced from either synthetic genes or from cDNA isolated from leeches.…”