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PubMedDate not supplied

[Determination of the antigenic composition of the hemagglutinins of influenza viruses H1N1].

A comparative immunological analysis of the antigenic composition of hemagglutinins (HA) of influenza A viruses isolated from man and animals was done by the method of specific adsorption of immunoglobulin fractions recovered from sera of rabbits and rats immunized with influenza H1N1 viruses isolated in 1930-1948. A single determinant which remains antigenically stable from 1930 up to the present time was found in HA of human influenza H1N1 viruses (1933-1957, 1977-1981), swine virus A/Swine/Iowa/30, and whale virus A/whale/Pacific Ocean/8/76. Animal influenza viruses A/swine/Iowa/30 and A/whale/Pacific Ocean/8/76 have in their HA one more determinant contributing to the similarity of the swine virus with human A/PR8/34 virus, and the whale virus with human A/W.Smith/33 virus.

Adsorption↗
PubMedDate not supplied

[The genetic differentiation of mammalian taxa: their assessment by biochemical genetic markers].

A review of data on genetic differentiation of mammalian taxa has been made on the basis of estimating the percent of fixed gene differences (PFD). The results substantiate the existence of evident differences in the scale of genetic divergence between taxa in different mammalian orders. Among smaller mammals (marsupials, insectivores, chiropterans, myomorph and sciuromorph rodents, african mole rats, and elephant shrews) interspecific differences within a genus involve the average of 25-40% of investigated loci. At the genetic level the value is 50-60%, whereas at the familial level the differences are beyond the resolution capacity of the method (PFD = 60-80%). Orders of larger mammals can be divided into two subgroups. One of them that includes carnivores, artiodactylans, and hystricomorph rodents is characterised by PFD values of 10-14%, 30-50%, and 69-70% at respective levels. The other subgroup composed of proboscideans, primates, pinnipeds, and toothed whales, has a low level of genetic divergence expressed by PFD values of 0-3%, 7-36%, and 50-60% at species, generic and familial levels, respectively. Insufficiency of data on baleen whales and perissodactyls does not allow to cluster them ultimately with any of these groups. There are three possible, but not necessarily alternative, causes for the observed differences in genetic divergence: 1) over-ranking of genera in larger mammals; 2) different paleontological age of orders; 3) unequal rates of molecular evolution.

Animals↗
PubMed1993

Structural studies on the loggerhead sea turtle (Caretta caretta) myoglobin.

The primary structure of myoglobin from the loggerhead sea turtle (Caretta caretta) has been determined; the protein consists of 153 amino acid residues. The ferric loggerhead sea turtle myoglobin has been crystallized in a form suitable for X-ray structural investigations. The crystals were grown at pH 8.0, in 0.05 M tris/HCl buffer, using 3.2 M ammonium sulfate as precipitating agent, at 4 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell constants a = 37.2 A, b = 61.1 A, c = 75.2 A (one molecule, 17,000 M(r), in the asymmetric unit). A molecular replacement solution was found for the loggerhead sea turtle myoglobin crystals using sperm whale myoglobin structure as search model. The R-factor value, after molecular replacement, is 0.387, for the data in the 15-3.3 A resolution range. The results here reported are the basis for the first X-ray crystallographic investigation on a reptile myoglobin, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian (i.e. sperm whale) myoglobins.

Amino Acid Sequence↗
PubMed1996

Quantitative analysis of T cell activation: role of TCR/ligand density and TCR affinity.

(B6 X A)F1 mice were immunized with sperm whale myoglobin, and T cell clones and hybridomas were generated. Hybridoma 74a.e9 was specific for the sperm whale myoglobin 67-79 peptide and could be partially activated by a peptide analogue, equine myoglobin with a natural 74G substitution. Using this hybridoma in T cell activation assays, we studied the effects of varying the avidity of the TCR for its ligand, the concentration of MHC:peptide complex on the APC, and the density of TCR on the surface. Varying ligand concentration on the surface of the APC, the TCR avidity, or the density of TCR on the T cell were equally important parameters in driving T cell activation. The mouse myoglobin (74T) analogue, however, acted as an antagonist to the T cell response. Its effectiveness was also partially determined by its ability to bind to MHC. By independently altering each of these variables and following T cell activation, we describe the interrelationships among these three components (MHC:peptide:TCR) that control the activation of the T cell.

Amino Acids↗
PubMed1998

Comparative kinematics and hydrodynamics of odontocete cetaceans: morphological and ecological correlates with swimming performance.

Propulsive morphology and swimming performance were compared for the odontocete cetaceans Delphinapterus leucas, Orcinus orca, Pseudorca crassidens and Tursiops truncatus. Morphological differences were apparent among the whales. The general body contour and low-aspect-ratio caudal flukes of D. leucas indicated that this species was a low-performance swimmer compared with the other species. Propulsive motions were video-taped as animals swam steadily in large pools. Video tapes were analyzed digitally using a computerized motion-analysis system. Animals swam at relative velocities ranging from 0.4 to 2.4 body lengths s-1. The stroke amplitude of the flukes decreased linearly with velocity for D. leucas, but amplitude remained constant for the other species. Tail-beat frequencies were directly related to relative swimming velocity, whereas the pitch angle of the flukes was inversely related to relative swimming velocity. Unsteady lifting-wing theory was used with regression equations based on kinematics to calculate thrust power output, drag coefficients and propulsive efficiency. Compared with other species, O. orca generated the largest thrust power (36.3 kW) and had the lowest drag coefficient (0.0026), whereas T. truncatus displayed the largest mass-specific thrust power (23.7 W kg-1) and P. crassidens had the highest efficiency (0.9). D. leucas did not swim as rapidly as the other species and had a comparatively higher minimum drag coefficient (0.01), lower mass-specific thrust power (5.2 W kg-1) and lower maximum efficiency (0.84). Minimum drag coefficients were associated with high swimming speeds, and maximum efficiencies corresponded with velocities in the range of typical cruising speeds. The results indicate that the kinematics of the propulsive flukes and hydrodynamics are associated with the swimming behaviors and morphological designs exhibited by the whales in this study, although additional factors will influence morphology.

Journal Article↗
PubMed1998

Comparative kinematics and hydrodynamics of odontocete cetaceans: morphological and ecological correlates with swimming performance.

Propulsive morphology and swimming performance were compared for the odontocete cetaceans Delphinapterus leucas, Orcinus orca, Pseudorca crassidens and Tursiops truncatus. Morphological differences were apparent among the whales. The general body contour and low-aspect-ratio caudal flukes of D. leucas indicated that this species was a low-performance swimmer compared with the other species. Propulsive motions were video-taped as animals swam steadily in large pools. Video tapes were analyzed digitally using a computerized motion-analysis system. Animals swam at relative velocities ranging from 0.4 to 2.4 body lengths s-1. The stroke amplitude of the flukes decreased linearly with velocity for D. leucas, but amplitude remained constant for the other species. Tail-beat frequencies were directly related to relative swimming velocity, whereas the pitch angle of the flukes was inversely related to relative swimming velocity. Unsteady lifting-wing theory was used with regression equations based on kinematics to calculate thrust power output, drag coefficients and propulsive efficiency. Compared with other species, O. orca generated the largest thrust power (36.3 kW) and had the lowest drag coefficient (0.0026), whereas T. truncatus displayed the largest mass-specific thrust power (23.7 W kg-1) and P. crassidens had the highest efficiency (0.9). D. leucas did not swim as rapidly as the other species and had a comparatively higher minimum drag coefficient (0.01), lower mass-specific thrust power (5.2 W kg-1) and lower maximum efficiency (0.84). Minimum drag coefficients were associated with high swimming speeds, and maximum efficiencies corresponded with velocities in the range of typical cruising speeds. The results indicate that the kinematics of the propulsive flukes and hydrodynamics are associated with the swimming behaviors and morphological designs exhibited by the whales in this study, although additional factors will influence morphology.

Animals↗
PubMed2000

Trans-substitution of the proximal hydrogen bond in myoglobin: II. Energetics, functional consequences, and implications for hemoglobin allostery.

The trans-substituted histidine to glycine mutant of sperm whale myoglobin (H93G Mb) is used to study energetics of proximal hydrogen bonding, proximal ligand-heme interactions, and coupling to distal ligand binding. Comparison of mono- and dimethylimidazole structural isomers shows that the hydrogen bond between the proximal ligand and the neighboring Ser92 hydroxyl (position F7) is stabilizing. The range of hydrogen bond stabilities measured here for different distal ligand complexes ranges from -0.7 kcal/mol (monomethylimidazole isomers to MbCO) to -4.1 kcal/mol (dimethylimidazole isomers to MbCN). This range of hydrogen bond stabilities, which is similar to that seen in protein mutagenesis unfolding studies, demonstrates the high sensitivity of the hydrogen bond to modest structural perturbations. The degree to which the 2-methyl group destabilizes proximal ligand binding is found to depend inversely on the total electronic spin. For monomethylimidazole proximal ligands, distal ligand binding weakens the proximal hydrogen bond compared to deoxyMb. Surprisingly, this trend is largely reversed for the dimethylimidazole proximal ligands. These results demonstrate strong coupling between the proximal protein matrix and distal ligand binding. These results provide an explanation for the strong avoidance of hydrogen bonding residues at position F7 in hemoglobin sequences.

Animals↗
PubMed2002

Characterization and peroxidase activity of a myoglobin mutant containing a distal arginine.

The spectroscopic, conformational, and reactivity characteristics of the T67R variant of sperm whale myoglobin have been studied to assess the effects of introducing an arginine residue into the distal side of this protein, as occurs in the active site of heme peroxidases. The overall circular dichroism (CD) and NMR spectroscopic properties of various derivatives of the protein are little affected by the mutation. The mutant contains a high-spin ferric ion with a water molecule as the sixth ligand, which exhibits slightly enhanced acidity (pK(a)=8.43+/-0.03) with respect to the corresponding derivative of wild-type myoglobin (pK(a)=8.60+/-0.04). The presence of the distal arginine increases the affinity of the Fe(III) center for azide (K=(6.0+/-0.5)x10(4) M(-1)) and decreases that for imidazole (K=12.0+/-0.2 M(-1)), with respect to the wild-type protein (K=(5.0+/-0.1)x10(4) and 24.7+/-0.7 M(-1), respectively). The peroxidase activity of T67R and wild-type myoglobins has been studied with a group of phenolic substrates related to tyrosine. The mutant exhibits an increased rate of reaction with hydrogen peroxide (k=1550+/-10 versus 760+/-10 M(-1) x s(-1)) and a generally increased peroxidase activity with respect to wild-type myoglobin. Relaxation measurements of proton nuclei of the phenolic substrates in the presence of either the T67R variant or the wild-type protein show that binding of these molecules occurs at distances of 8-10 A from the iron center, that is, close to the heme pocket, except for p-cresol, which can approach the heme more closely and, therefore, probably enter into the distal cavity.

Animals↗
PubMed1986

Microanatomy of the renicule of Balaena mysticetus.

Renicules from twelve bowhead whales (Balaena mysticetus) were examined utilizing light, scanning electron, and transmission electron microscopes. The basic organization of the renicule into capsule, cortex, sporta perimedullaris, medulla, and calyx is described. Despite less than perfect preservation resulting from environmental and logistical conditions at the collecting sites, it has been possible to document the basic microstructure of most components of the renicule of this endangered species. Several unusual features were observed. The absence of smooth muscle fibers (other than in vessel walls) from the capsule, sporta perimedullaris, and calyx wall is a departure from what is reported in other cetaceans as is the consistent presence of arcuate arteries in the substance of the sporta perimedullaris. Large subcapsular veins are present but do not appear to represent connecting elements in an alternative venous return through capsular and interrenicular veins. Elastic fibers are seen only in the sporta perimedullaris and the calyx wall, whereas reticular fibers are most abundant in the medullary stroma. Finally, enlarged cells with clear cytoplasm are seen in the tunica media of the glomerular afferent arterioles extending a variable, but always considerable, distance toward the interlobular arteriole. These cells are presumed to represent an extended array of the epithelioid cells common in the afferent arterioles of the juxtaglomerular apparatus of other mammalian kidneys.

Animals↗
PubMed1995

Generation of antigen-specific CD4+ T cell lines from naive precursors.

The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.

Animals↗
PubMed1994

Interleukin-6 antisense deoxyoligonucleotides inhibit bone resorption by giant cells from human giant cell tumors of bone.

The effects of antisense constructs to IL-6 on the bone-resorbing capacity of purified giant cells from giant cell tumors of bone were examined to further define the role of IL-6 in human osteoclastic bone resorption. In addition, we wanted to determine the utility of antisense constructs to cytokines produced by osteoclasts as probes to identify the molecular events responsible for the bone-resorptive process. Giant cells were cultured on sperm whale dentin for 24 h in the presence of fluoresceinated antisense or scrambled antisense deoxyoligonucleotides complementary to IL-6 mRNA. The giant cells actively incorporated these oligonucleotides, as evidenced by their intense fluorescence. The number of resorptive lacunae formed and the area of the dentin resorbed were significantly decreased in cultures of giant cells treated with antisense IL-6 constructs compared with control cultures treated with scrambled antisense constructs to IL-6 (60 +/- 13 versus 12 +/- 6 lacunae and 1.2 +/- 0.3 versus 0.26 +/- 0.1 x 10(5) microns2). IL-6 levels in conditioned media from giant cell cultures treated with IL-6 antisense constructs were fourfold lower than those in control media obtained from giant cells treated with scrambled antisense constructs to IL-6. These data confirm the capacity of IL-6 antisense oligomers to block IL-6 production by these cells. These observations show that IL-6 plays an important role in the bone-resorptive process of human osteoclasts and suggest that antisense constructs to cytokines produced by bone cells may be useful for determining the molecular events occurring during bone resorption.

Animals↗
PubMed1981

Arterial circulation of the spinal cord and brain in the Monodontidae (order Cetacea).

In this paper we document retial supply of the spinal cord and describe the arterial vascular pattern of the brain in the whale family Monodontidae. Observations are based on gross dissections of four brains, two each of Monodon monoceros and Delphinapterus leucas, and one spinal cord from M. monoceros. Vessels of the spinal cord arise from extradural retia in the neural canal. Arteries originating from the retia penetrate the dura between successive spinal roots (mainly ventral) and not in association with them, unlike radicular arteries of other mammals. Also, these vessels are uniformly distributed and contribute equally to a plexus surrounding the cord. An A. radicularis magna is not present, and neither are distinct anterior or posterior spinal arteries. Circulation to the brain is effected by two pairs of arteries originating from intracranial retia. The rostral pair supplies most of the forebrain (prosencephalon), whereas the more caudal pair supplies mainly the midbrain (mesencephalon) and hindbrain (rhombencephalon). The circulatory pattern is characterized by 1) complete independence of anterior cerebral arteries (no anastomoses); 2) extensive cortical supply by the anterior choroidal arteries; 3) absence of subdural communicating vessels between rostral and caudal trunks; 4) union of caudal trunks to form a small basilar artery; and 5) absence of vertebral arteries and hence of a vertebral basilar system. There are some obvious differences between subdural arteries in the Monodontidae and those in other mammals; however, their general patterns of distribution are similar, and we suggest that most of the vessels, at least in the cranium, are homologous.

Animals↗
PubMed1994

Microscopy of vascular architecture and arteriovenous communications in the spleen of two odontocetes.

The red pulp of the spleens of the short-finned pilot whale (Globicephala macrorhynchus) and the Pacific bottle-nosed dolphin (Tursiops truncatus gilli) (Odontoceti) were examined by light and electron microscopy and found to comprise two venous layers, an inner and an outer. The inner layer is homologous to the intermediate zone (IZ) of primitive-type mammalian spleens and contains sinusoids consisting of endothelial cells and a thin layer of extracellular deposits. Its vascular structure is unclear. The venous vessels of this layer eventually communicate with veins of the perivenous outer layer. The perivenous layer contains veins of various sizes, interstitial elements, and trabeculae. It is filled with blood cells, particularly plasma cells, but no myeloid cells. The perivenous layer (PVL) is homologous to the red pulp of common mammalian spleens but shows signs of involution. The white pulp gives origin to arterial terminals that end in the red pulp, where they communicate directly with the sinusoidal veins producing a closed circulation. The arterial terminals do not show ellipsoids. The presence of an IZ with a closed circulation and the involution of the red pulp makes the spleen of Odeontoceti another example of a mammalian spleen of the primitive type that has been altered by the evolutionary process. Vascular remodelling of the spleen of Odontoceti seems to follow the pattern noted in the spleens of nonmammalian vertebrates.

Animals↗
PubMed1998

Relationships between bone morphology and bone elastic properties can be accurately quantified using high-resolution computer reconstructions.

It would be advantageous if the mechanical properties of trabecular bone could be directly inferred from stereomorphometric parameters. For that purpose, apparent density and mean intercept length, as measures of bone mass and directionality (fabric), are commonly correlated with the elastic characteristics of bone samples, as determined in compression tests. However, complete and accurate relationships have not yet been established in this way. This may be due not only to the occurrence of artifacts in both the stereomorphometric and the mechanical assessments but also to an inherent inadequacy of mean intercept length in characterizing the full mechanical significance of bone architecture or nonhomogeneities in trabecular tissue properties not accounted for in stereomorphometry. In this study, we introduce a computer modeling approach allowing these biases to be eliminated. With use of high-resolution three-dimensional computer reconstructions of trabecular bone specimens for stereomorphometry and for microstructural finite element models to simulate mechanical tests, unbiased comparisons become feasible. The purpose was to investigate if accurate and complete relationships can be established in this way. Four different fabric measures were considered: mean intercept length and three volume-based ones. Compliance matrices were calculated from fabric tensors, with use of the mathematical relationship proposed by Cowin for 29 vertebral whale-bone specimens. These were correlated with the compliance constants determined directly from the microstructural finite element model simulation. The nine orthotropic elastic constants of all 29 specimens were well predicted from their stereomorphometric fabric and volume fraction values, with correlation coefficients ranging from R2adj = 0.9934 to 0.9963. When individual compliance components were considered (1/Ei, 1/Gij, or -v[ij]/Ei), correlation coefficients ranged from R2adj = 0.924 to 0.982. All four fabric measures performed equally well. It is concluded that volume fraction and fabric measures correlate highly with the apparent elastic properties of bone samples, provided that anisotropy and nonhomogeneity in the elastic properties of the trabecular tissue itself have negligible effects on the apparent properties. Whether this is true for bone in general remains to be seen, as only a subset was analyzed here. These methods, however, can be valuable in similar assessments of other subsets.

Animals↗
PubMed1994

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.

Amino Acid Sequence↗
PubMed1994

Stabilization of myoglobin by multiple alanine substitutions in helical positions.

We have carried out a series of multiple Xaa-->Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed Tm (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.

Alanine↗
PubMed1997

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.

Amino Acid Sequence↗
PubMed2002

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme.

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.

Animals↗