Camel myoglobin

1. Crystalline myoglobin was prepared from camel heart muscle. 2. A method was developed for the isolation of myoglobin that employs molecular-sieve chromatography. 3. Analytical chromatography of the camel myoglobin on a molecular-sieve column and on two types of ion-exchange columns gave in each case a single elution band, which accounted for better than 98% recovery and showed that the product was free from haemoglobin. 4. The iron content on a dry weight basis was 0.308%. This value corresponds to a molecular weight of 18100. 5. The spectra of acidic ferrimyoglobin, basic ferrimyoglobin and ferrimyoglobin cyanide were measured. 6. The pK(a) of the dissociation of the haem-bound water molecule in acidic ferrimyoglobin was 8.53 at 25 degrees . 7. Conclusions are drawn about the charge on the surface of the camel ferrimyoglobin molecule as compared with horse and sperm-whale ferrimyoglobins.     

Large-scale oscillatory motions in myoglobin

Elastic and inelastic components of neutron scattering from lyophilized myoglobin were measured at wavevector transfers, Q of 1.0, 1.4, 1.85, and 3.0 Å^-1 and at temperatures of 77 and 298 K. The elastic scattering determines an overall effective mean square atomic displacement which was employed in a simple harmonic Langevin formalism to obtain effective force constants describing molecular deformations. These force constants and the inelastic data are compatible with alpha helices as a vibrating species at room temperature, and with amino acid residues as the vibrating entities at 77 K. The present measurements are consistent with recent molecular dynamics calculations.     

Dioxygen replacement reaction in myoglobin

The replacement reaction of myoglobin (Mb), MbCO + O2 leads to MbO2 + CO leads to MbCO + O2, has been studied with flash photolysis in the temperature range from 140 to 320 K and the time range from 2 mus to 200 s. In a fraction of the Mb, the photodissociated CO remains within the protein; rebinding is not affected by the presence of O2 and occurs with rates that are identical with the ones observed earlier in solvents containing only CO. In the remaining fraction CO migrates into the solvent and Mb combines preferentially with oxygen. The rate of the subsequent replacement of O2 by CO permits calculation of the oxygen dissociation rate ko2; ko2 has been determined from 260 to 320 K. The measurements support a multibarrier model.     

Relaxations and fluctuations in myoglobin

One major goal of biological physics is the discovery and understanding of the concepts and laws that govern biomolecules, in particular proteins. Since there exist at least 10(5) different proteins, the choice of a suitable prototype is necessary. Myoglobin (Mb) has for many years played the role of such a prototype. It appears to be simple enough so that many of its properties can be understood, yet it is complex enough to display many of the fascinating characteristics of biomolecules. One major achievement in the study of any protein would be the establishment of convincing connections among structure, kinetics, energy landscape, dynamics, and function. We believe that this goal has not yet been reached in any protein, but the present knowledge of Mb gives some hope that the end is near in this case. Here, we sketch some of the results that have been obtained in the past 50 or more years in the research on Mb, obtained by an army of investigators.     

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.     

Equilibrium fluctuations in myoglobin and lysozyme

The angular dependencies of inelastic intensities of Rayleigh scattering of Moessbauer radiation were measured for myoglobin and lysozyme (in the hydration range h = 0.05-0.7). The data were fitted within the framework of model, when two types of intraglobular motions were taken into account: individual motions of small side-chain groups and cooperative motions of segments. The best agreement with the experiment at h > 0.05 was obtained when individual motions of small groups together with the cooperative motions of alpha-helices and beta-sheets for lysozyme, and alpha-helices for myoglobin were considered. At further hydration (h = 0.45), mean-square displacements (x2) of both types of motions strongly increase with the increase in hydration degree, while the motions with a large correlation radius (not less than macromolecule radius) remain nearly the same as for h = 0.05. The results of the study of the radial distribution function deduced by Fourier-transform from the diffuse x-ray measurements together with RSMR data allow one to conclude that the water during protein hydration competes with the intramolecular hydrogen bonds, loosens the protein and increases the internal dynamics. Concurrently, water arranges the ordering of macromolecule, which takes the native structure at h = 0.4-0.7. The analysis of auto and cross-correlation functions of bending fluctuations of alpha-helices in the large domain of lysozyme performed by molecular dynamics allows one to come to the final conclusion that it is the difference in the structural organization of myoglobin and lysozyme and not the presence of SS-bonds in lysozyme macromolecule that is responsible for different structural fluctuations in these proteins.     

Structural dynamics of myoglobin

Conformational fluctuations have been invoked to explain the observation that the diffusion of small ligands through a protein is a global phenomenon, as suggested (for example) by the oxygen induced fluorescence quenching of buried tryptophans. In enzymes processing large substrates, a channel to the catalytic site is often seen in the crystal structure; on the other hand in small globular proteins, it is not known if the cavities identified in the interior space are important in controlling their function by defining specific pathways in the diffusion to the active site. This point is addressed in this paper, which reports some relevant results obtained on myoglobin, the hydrogen atom of molecular biology. Protein conformational relaxations have been extensively investigated with myoglobin because the photosensivity of the adduct with CO, O2 and NO allows us to follow events related to the migration of the ligand through the matrix. Results obtained by laser photolysis, molecular dynamics simulations, X-ray diffraction of intermediate states of wt type and mutant myoglobins are briefly summarized. Crystallographic data on the photochemical intermediate of a new triple mutant of sperm whale myoglobin (Mb-YQR) show, for the first time, the photolyzed CO* sitting in one of the Xe-binding cavities, removed from the heme group. These results support the viewpoint that pre-existing 'packing defects' in the protein interior play a major role in controlling the dynamics of ligand binding, including oxygen, and thereby acquire a survival value.     

Cold denaturation of myoglobin

The stability of the structure of sperm whale metmyoglobin has been studied in various solutions, in the temperature range -8 degrees C to 100 degrees C, by scanning microcalorimetry, light absorption, circular dichroism, nuclear magnetic resonance spectroscopy and viscosimetry. It has been shown that in 10 mM-sodium acetate solutions (pH 3.5 to 3.9) the protein molecule undergoes a reversible conformational transition into a non-compact disordered state not only when the solution is heated above room temperature but also when it is cooled. In this state the protein does not have a tertiary structure, although it retains some residual ellipticity, which may be caused by the fluctuating alpha-helical conformation of the unfolded polypeptide chain. The disruption of the native protein structure both on cooling (cold-denaturation) and on heating (heat-denaturation) proceeds in an "all-or-none" manner, with a significant and similar increase of the protein heat capacity, but with inverse enthalpic and entropic effects: the enthalpy and entropy of the protein molecule decrease during cold-denaturation and increase during heat-denaturation.     

The tautomeric state of histidines in myoglobin

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.     

Molecular dynamics simulation of hydration in myoglobin

This study was carried out to evaluate the stability of the 89 bound water molecules that were observed in the neutron diffraction study of CO myoglobin. The myoglobin structure derived from the neutron analysis was used as the starting point in the molecular dynamics simulation using the software package CHARMM. After solvation of the protein, energy minimization and equilibration of the system, 50 ps of Newtonian dynamics was performed. This data showed that only 4 water molecules are continously bound during the length of this simulation while the other solvent molecules exhibit considerable mobility and are breaking and reforming hydrogen bonds with the protein. At any instant during the simulation, 73 of the hydration sites observed in the neutron structure are occupied by water. © 1995 Wiley-Liss, Inc.     

S-nitrosylation-induced conformational change in blackfin tuna myoglobin

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.     

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.