1H-n.m.r. and c.d. studies of haem orientational disorder in sperm-whale myoglobin and human haemoglobin.
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ABSTRACT:
SUBMITTER: Aojula HS
PROVIDER: S-EPMC1148933 | biostudies-other | 1988 Mar
REPOSITORIES: biostudies-other
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Project description:Ligand-binding kinetics of native and reconstituted sperm-whale myoglobin were studied in relation to haem orientational disorder by rapid kinetic methods. In addition, native yellow-fin-tuna myoglobin with significant amount of haem disorder was also used. The O2 dissociation and association rates were found for the proteins with different degrees of haem disorder, and these results suggest that the isomers are characterized by almost identical kinetic parameters. Rates of CO recombination after photolysis were also identical for the two orientational isomers. The results clearly indicate that the rotation of the haem about the alpha-gamma meso axis has little or no effect on the ligand-binding properties of these myoglobins.
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Project description:1. Crystalline myoglobin was isolated from the skeletal muscle of the finback whale and fractionated, in its cyanmet form, into nine components (I-IX) by chromatography on CM-cellulose. Also in the cyanmet form, it was resolved into six components by electrophoresis on starch gel. Correspondence between the electrophoretic and chromatographic components was determined, and interconversion between components revealed by chromatography and electrophoresis. 2. The chromatographic myoglobin components were homogeneous in the ultra-centrifuge. Molecular weights of certain components were determined by means of sedimentation equilibrium and by gel filtration on Sephadex G-100. Values from these two methods corresponded to the minimum molecular weight calculated from the iron content. 3. The spectral properties of the chromatographic components were investigated in the visible and the ultraviolet ranges. 4. The major components of finback-whale myoglobin and sperm-whale myoglobin showed almost identical spectral, electrophoretic and chromatographic behaviours, but had different infrared spectra. The infrared spectra of the corresponding apoproteins were almost identical. 5. Rabbit antisera to sperm-whale myoglobin component X cross-reacted with finback-whale myoglobin components V, VI and VII only about 30%. 6. The major chromatographic components of finback-whale myoglobin have identical amino acid compositions. The polypeptide chain contains 151 amino acid residues and its molecular weight is 17504. 7. The N-terminal end of the chain is: [Formula: see text] Amino acids released from myoglobin by the action of carboxypeptidase A at different intervals were determined.
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Project description:In this article we use the perturbed matrix method and an extended molecular dynamics sampling of the carbon monoxide (CO) in the myoglobin distal pocket to characterize the CO vibrational spectrum and hence to relate its spectroscopic features with the atomic-molecular behavior. Results show the accuracy of the method employed and confirm the assignment of the spectroscopic B1 and B2 states proposed by Lim et al.
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Project description:We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.
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Project description:Degradation of the haem of haemoglobin (used as a chemical probe of the haem-protein relationship), suggests that reconstituted human haemoglobin contains significant haem disorder. This results from the insertion of haem into globin with an orientation 180 degrees different from the natural orientation. Haem disorder also slowly occurs in methaemoglobin solutions.
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Project description:The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.
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Project description:1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin.
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Project description:Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.
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Project description:The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.
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Project description:Immunoadsorbents that were potentially suitable for the fractionation of antibodies to myoglobin were prepared by coupling myoglobin to aminoethylcellulose, bromoacetylcellulose, CM-cellulose, an ethylene-maleic anhydride co-polymer and Sepharose 4B. A comparison of the properties of the preparations indicated that Sepharose-myoglobin was superior, as an immunoadsorbent, to the other conjugates. Elution of adsorbed antibodies with m-propionic acid, pH2.5, gave yields of precipitable antibody higher than those achieved with other dissociating solvents. Propionic acid eluted 60-100% of the antibody adsorbed by Sepharose-myoglobin, and 60-90% of the eluted protein was precipitable antibody.
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