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PubMed1993

Conformational deformation in deoxymyoglobin by hydrostatic pressure.

Pressure effect on the equilibrium conformation in sperm whale deoxymyoglobin and its volume fluctuation are studied by the normal mode analysis and strain tensor analysis. The pressure-induced deformation of interhelix regions are found to be remarkably more compressed than the other parts of the molecule. The intrahelix compressibility is shown to be relatively small. We also calculate the compressibility of the three hydrophobic clusters, located at the bottom, distal, and proximal side of the heme. Its value is found to decrease in the indicated order. The average compressibility of these hydrophobic clusters is less than the average interhelix compressibility, even though there are large cavities in these clusters. In order to study overall deformation, we define a linear compressibility and calculate it for all pairs of C alpha atoms. The pressure-induced deformation is observed to be heterogeneous also in this analysis. The calculated root-mean-square displacement of the constituent atoms in the equilibrium conformation at 1,000 atm from those at 0 atm is 0.12 A, which is much smaller in magnitude than the average value of the atomic fluctuations at room temperature. In the water solvent, the volume excluded by the protein molecule in the equilibrium conformation is reduced by 0.9%, when the pressure is raised from 0 to 1,000 atm. The calculated magnitude of the root-mean-square volume fluctuation is 0.3% of the excluded volume at room temperature. The square of the volume fluctuation is given as a sum of contributions from individual normal modes. Contributions from low frequency normal modes are found to dominate over those from higher frequency normal modes. The estimated value of the isothermal compressibility of deoxymyoglobin is 9.37 x 10(-12) cm2/dyn.

Animals↗
PubMed1992

Matrix-assisted laser desorption using a fast-atom bombardment ion source and a magnetic mass spectrometer.

A conventional fast-atom bombardment (FAB) ion source was used to achieve matrix-assisted laser desorption (MALD) in a high-mass, double-focusing, magnetic mass spectrometer. The pulsed ion signals generated by irradiation of a mixture of sample and matrix (2,5-dihydroxybenzoic acid) with either a XeF excimer laser (353 nm) or a nitrogen laser (337 nm) were recorded with a focal-plane detector. A resolution (full-width at half maximum) of 4500 was achieved at m/z 1347.7 (the peptide substance P), 2500 for CsI cluster ions at m/z 10,005.7, and 1250 for the isotope cluster of the small protein cytochrome c (horse) [M+H]+ = m/z 12,360 (average). Sensitivity is demonstrated with 11 fmol of substance P. A survey scan is taken to locate the m/z of the sample molecular ion. The segment that contains the sample can then be integrated for a longer time to produce a better signal-to-noise ratio. In addition to higher sensitivity and lower matrix interference, the advantage of MALD over FAB is the former's lower susceptibility to the presence of salts, and competition between hydrophobic and hydrophilic components of a mixture. This feature is demonstrated by the complete MALD spectrum of a crude partial tryptic digest of sperm-whale apomyoglobin, containing 24 peptides, representing the entire sequence of this protein.

Animals↗
PubMed1994

Requirement of a second oxidation equivalent for ferryl oxygen transfer to styrene in the epoxidation catalyzed by myoglobin-H2O2.

Ferric myoglobin (Mb) is oxidized by H2O2 to a ferryl (FeIV = O) species and a protein radical, whereas ferrous Mb is similarly oxidized to the ferryl species without the protein radical. The protein radical is unstable and decays within 5 min, but the ferryl species is stable for more than 1 h. Previous studies have shown that styrene is oxidized to styrene oxide and benzaldehyde by ferric Mb and H2O2. We demonstrate here that the ferryl species produced from ferrous equine Mb and H2O2 does not epoxidize styrene. Furthermore, the EPR signal intensity of the protein radical formed from ferric equine Mb and the ability to oxidize styrene decrease in parallel as a function of the time separating the addition of H2O2 and styrene. The ability to oxidize styrene as a function of time after addition of H2O2 is lost much more rapidly with the H64V/K102Q/Y103F/Y146F/Y151F mutant of sperm whale Mb than with the native protein or the Y146F/Y151F mutant. The results indicate that styrene epoxidation requires a two-electron oxidized species of Mb in which the ferryl (FeIV = O) complex is coupled to a protein or highly transient (undetectable) porphyrin radical. Benzaldehyde formation appears to be catalyzed by the same oxidizing species. Styrene oxidation thus differs from linoleic acid oxygenation, which is catalyzed by the ferryl species alone.

Animals↗
PubMed1995

Infrared analysis of ligand- and oxidation-induced conformational changes in hemoglobins and myoglobins.

Effects of the binding of O2 and CO to heme iron (II) of deoxy forms and of the oxidation of deoxy forms to aquoiron (III) complexes on the infrared spectra of hemoglobins and myoglobins have been examined. Spectra were measured for aqueous solutions 3-4 mM in heme of human, bovine, and equine hemoglobins and sperm whale, bovine, and equine myoglobins in 10 mM sodium phosphate buffer, pH 7.4, at 20 degrees C. All ligand binding and oxidation reactions resulted in similar spectral shifts in the region 1665 to 1670 cm-1, a portion of the amide I region assignable to beta-turn structure. There were no other significant changes in the amide I region, a finding consistent with no other alterations in secondary structure. The major bands near 1655 cm-1 associated with alpha-helices were consistently at 2 cm-1 lower wavenumber for myoglobins than for hemoglobins. The changes in solution infrared spectra observed in this study may result at least in part from conformational changes at the FG corner associated with movements of F and E helices that have been noted previously in crystal structures.

Animals↗
PubMed1996

Protein radicals in myoglobin dimerization and myoglobin-catalyzed styrene epoxidation.

Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P. R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (FeIV = O) oxygen transfer with retention of stereochemistry and incorporation of an oxygen from H2O2, and (b) protein peroxy radical cooxidation with loss of stereochemistry and incorporation of an oxygen from O2. As shown here, cis-beta-methylstyrene is preferentially oxidized to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4:1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide ratio increase in proportion to the cis-beta-methylstyrene concentration. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (approximately 30%) being obtained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidation equivalents utilized for protein oligomerization and styrene oxidation account for the available H2O2. Previous studies have shown that His-64 is important for protein-mediated olefin cooxidation and Tyr-151/Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants have now been prepared and the W14F, but not W7F, mutation shown to modestly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxide. Neither tryptophan mutation alters dimer formation. Dimer formation is modestly increased rather than decreased by styrene, suggesting that styrene cooxidation and dimerization do not compete. The results indicate that (a) cis-beta-methylstyrene cooxidation and protein dimerization, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl epoxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly nonequilibrating, radicals mediate olefin cooxidation and protein dimerization.

Animals↗
PubMed2001

Molecular evolution of leptin.

Leptin, a hormone produced mainly by adipocytes, is involved in the regulation of food intake, metabolism, and reproduction. The objective of this study was to determine the evolutionary relationships of leptin genes. Partial nucleotide sequences of leptin were cloned and sequenced from six mammalian species: large hairy armadillo (Chaetophractus villosus), rabbit (Oryctolagus cuniculus), big brown bat (Eptesicus fuscus) [corrected], striped skunk (Mephitis mephitis), raccoon (Procyon lotor), and beluga whale (Delphinapterus leucas). The PUZZLE program was used to construct maximum-likelihood trees. Our phylogenetic analysis shows that the grouping of these new mammalian sequences with those currently available in GenBank respect the evolutionary relationships generally accepted for mammals. However, when leptin sequences for chicken and turkey are included in the analysis, these are found to group with mouse and rat leptins. Chicken and mouse leptins are 95% identical. However, when mouse is compared with closer relatives, such as rabbit or bat, identities are approximately 80%. A comparison of extant and ancestral leptin sequences suggests that convergent or parallel evolution is the most plausible hypothesis to explain the similarity between bird and rodent leptins.

Adipose Tissue↗
PubMed1995

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1996

Crystal structures of CO-, deoxy- and met-myoglobins at various pH values.

The distal histidine residue, His64(E7), and the proximal histidine residue, His93(F8), in myoglobin (Mb) are important for the function of the protein. For example, the increase in the association rate constant for CO binding at low pH has been suggested to be caused by the protonation of these histidine residues. In order to investigate the influence of protonation on the structure of myoglobin, we determined the crystal structures of sperm whale myoglobin to 2.0 A or better in different states of ligation (MbCO, deoxyMb and metMb) at pH values of 4, 5 and 6. The most dramatic change found at low pH is that His64 swings out of the distal pocket in the MbCO structure at pH 4, opening a direct channel from the solvent to the iron atom. This rotation seems to be facilitated by conformational changes in the CD corner. The benzyl side-chain of Phe46(CD4), which has been suggested to be a critical residue in controlling the rotation of His64, moves away from His64 at pH 4 in the deoxyMb structure, allowing more free rotation of His64. Arg45(CD3) is also important for the dynamics of myoglobin, since it influences the pK(a) of His64 and forms a hydrogen bond lattice that hinders the rotation of His64 at neutral pH. This hydrogen-bond lattice disappears at low pH. Although His64 rotates out of the distal pocket in the MbCO structure at pH 4, leaving more space for the CO ligand, the Fe-C-O angle refines to about 130 degrees, the same as those at pH 5 and 6. In the MbCO structure at pH 4, significant conformational changes appear in the EF corner. The peptide plane between Lys79(EF2) and Gly80(EF3) flips about 150 degrees. The occupancy of this conformation in the MbCO structures increases with decreases in pH. On the proximal side of the heme, the bond between the heme iron atom and N(epsilon) of His93 remains intact under the experimental conditions in the MbCO and deoxyMb structures, but appears elongated in the metMb structure at pH 4, representing either a weakened bond or the breakage of the bond in some fraction of the molecules in the crystal.

Animals↗
PubMed1999

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Animals↗
PubMed2001

On the nature of conformational openings: native and unfolded-state hydrogen and thiol-disulfide exchange studies of ferric aquomyoglobin.

Native-state amide hydrogen exchange (HX) of proteins in the presence of denaturant has provided valuable details on the structures of equilibrium folding intermediates. Here, we extend HX theory to model thiol group exchange (SX) in single cysteine-containing variants of sperm whale ferric aquomyoglobin. SX is complementary to HX in that it monitors conformational opening events that expose side-chains, rather than the main chain, to solvent. A simple two-process model, consisting of EX2-limited local structural fluctuations and EX1-limited global unfolding, adequately accounts for all HX data. SX is described by the same model except at very low denaturant concentrations and when the bulky labeling reagent 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is used. Under these conditions SX can occur by a novel denaturant-dependent process. This anomalous behavior is not observed when the smaller labeling reagent methyl methanethiosulfonate is employed, suggesting that it reflects a denaturant-induced increase in the amplitudes of local structural fluctuations. It also is not seen in heme-free apomyoglobin, which may indicate that local openings are sufficiently large in the absence of denaturant to allow DTNB unhindered access. Differences in SX kinetics obtained using the two labeling reagents provide estimates of the sizes of local opening reactions at different sites in the protein. At all sequence positions examined except for position 73, the same opening event appears to facilitate exchange of both backbone amide and side-chain thiol groups. The C73 thiol group is exposed by a low-energy fluctuation that does not expose its amide group to exchange.

Amides↗
PubMed2002

Cross-correlation effects involving curie spin relaxation in methyl groups.

Cross-correlation effects arising in methyl protons due to the simultaneous presence of dipole-dipole, chemical shift anisotropy, and Curie spin relaxation mechanisms in paramagnetic systems are analyzed. We assess the potential of obtaining structural constraints from the cross-correlation of Curie spin relaxation with dipolar relaxation mechanisms among methyl proton spins. By theoretical analysis and numerical simulations we characterize the transfer functions describing the interconversion processes of different ranks of multispin order. The time dependence of these processes contains a new type of structural information, the orientation of the methyl C(3)-axis with respect to the electron center. Experimental confirmation is found for selected methyl groups in low spin Fe(3+) sperm whale myoglobin.

Animals↗
PubMed1999

Single-locus tests of microsatellite evolution: multi-step mutations and constraints on allele size.

We evaluate some common simulation procedures as well as a recently developed likelihood method used for testing hypotheses regarding microsatellite evolution. Results from simulated data revealed that the tests for the detection of multi-step mutations in general have some power, whereas tests for the presence of constraints on the repeat number have only very limited power. The tests were applied to population data obtained from nine different baleen whale populations. High agreement was found between results obtained using the simulation-based approach and results obtained using a likelihood ratio test. In four of the nine population samples the tests rejected the one-step mutation model. In two instances the significant deviation was due to excess of heterozygosity and in two instances to a reduced level of heterozygosity relative to the expectations under the stepwise mutation model. The former significant deviation was consistent with occasional multi-step mutations, whereas the latter may indicate the presence of constraints on the number of repeats.

Alleles↗
PubMed1982

Dissection of the molecular parameters for T-cell recognition of a myoglobin antigenic site.

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr-151 and Ala-144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity of T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavorable mode of folding, suggested a conformational dependency in T-lymphocyte recognition and was confirmed by additional studies employing peptide priming. Thus priming with either the 11- or 22-residue peptides afforded T-cells which proliferated to both of these two peptides, but not to intermediate-sized peptides. More importantly, peptide-primed cells did not proliferate on challenge in vitro with Mb. This indicated that the in vivo recognition (priming) was to the unfolded peptide and these cells will not therefore recognize native Mb in vitro. Tolerization of neonatal mice with native Mb, which was total for the native protein at the T-cell level, had no effect on in vivo recognition (priming) and in vitro proliferation due to the unfolded peptide. Conversely, tolerization by any unfolded peptide had little or no effect on in vivo recognition of, and proliferation to, native Mb. It was concluded that T-cell recognition of native proteins (or at least of Mb) is dependent on protein conformation. A peptide may not cause proliferation of native protein-primed T-cells even though it contains an antigenic site. Finally, macrophage presentation of protein antigens is not related to processing via fragmentation of antigen by macrophage.

Animals↗
PubMed1994

The use of microsatellites for genetic analysis of natural populations.

Microsatellites, tandemly repeated units of 2 to 5 bp are distributed throughout eukaryotic genomes. Length variation within microsatellites, caused by DNA slippage, can be amplified by PCR and used for DNA profiling. In this paper potential applications and limitations of this technique are discussed. Two case studies for pilot whales (Globicephala melas) and sheep (Ovis aries) exemplify the suitability of microsatellites for analyzing natural populations. Other currently available profiling techniques are compared to microsatellite analysis.

Animals↗
PubMed1994

Intermediate states in ligand photodissociation of carboxymyoglobin studies by dispersive X-ray absorption.

The ligand photodissociation of sperm whale carboxymyoglobin (MbCO) at low temperature (15K-100K) under extended illumination has been studied by X-ray Absorption Near Edge Structure (XANES) spectroscopy using the dispersive technique. XANES simulations through the multiple scattering (MS) approach allow one to interpret the spectroscopic data in structural terms, and to investigate the Fe site structure configurations of the states that follow the CO photodissociation as a function of temperature. The Fe site in the photoproduct is unbound, with an overall structure similar to the deoxy-form (Mb) of the protein. The Fe site structure changes from T < 30K(Mb*) to T > 50K (Mb**), revealing the existence of a slower unbound state Mb**. A model is proposed which includes the faster state (Mb*) as a planar porphyrin ring with a displacement of Fe from the heme plane of less than 0.3 A, and the slower state (Mb**) with a domed heme.

Absorptiometry, Photon↗
PubMed1985

Haem-apoprotein interactions detected by resonance Raman scattering in Mb- and Hb-derivates lacking the saltbridge His146 beta-Asp94 beta.

The dispersion of the depolarization ratio of oxidation- and spin-marker lines of sperm whale myoglobin derivatives (oxyMb, deoxyMb, ferric Mb-CN) and of ferric Hb-CN have been measured for different pH-values in the acid and alkaline region. No pH-dependence in the region above pH = 6.5 has been found. Below pH = 6.5, however, a significant pH-dependence of the oxyMb-oxidation marker line at 1,375 cm-1 exists. Additionally, a weak pH-dependence of the corresponding 1,355 cm-1 line of the deoxymyoglobin spectrum is observed. This effect can be explained assuming a titration of distal histidine, inducing a rupture of the ligand-imidazole H-bond in the case of oxymyoglobin. The pH-independent depolarization ratio disperson above pH = 6.5 in all systems investigated is explained by the lack of the haemoglobin saltbridge between His(HC3) beta and Asp(FG5) beta, which is essential for the cooperativity in the haemoglobin system.

Animals↗
PubMed1986

Optimal harvesting of a logistic population in an environment with stochastic jumps.

Dynamic programming is employed to examine the effects of large, sudden changes in population size on the optimal harvest strategy of an exploited resource population. These changes are either adverse or favorable and are assumed to occur at times of events of a Poisson process. The amplitude of these jumps is assumed to be density independent. In between the jumps the population is assumed to grow logistically. The Bellman equation for the optimal discounted present value is solved numerically and the optimal feedback control computed for the random jump model. The results are compared to the corresponding results for the quasi-deterministic approximation. In addition, the sensitivity of the results to the discount rate, the total jump rate and the quadratic cost factor is investigated. The optimal results are most strongly sensitive to the rate of stochastic jumps and to the quadratic cost factor to a lesser extent when the deterministic bioeconomic parameters are taken from aggregate antarctic pelagic whaling data.

Animals↗
PubMed1984

Monocyte-enriched cells on calcified tissues.

Monocyte-enriched human blood cells seeded on to sperm whale dentine and cultured for up to 20 days failed to produce any morphological signs of resorbtive activity, although multinucleate giant cells were formed. In contrast, preparations containing known osteoclasts derived from bone resorbed the same substrate within hours.

Animals↗