-
Biochemistry Sep 2023Proteins have undergone evolutionary processes to achieve optimal stability, increased functionality, and novel functions. Comparative analysis of existent and ancestral...
Proteins have undergone evolutionary processes to achieve optimal stability, increased functionality, and novel functions. Comparative analysis of existent and ancestral proteins provides insights into the factors that influence protein stability and function. Ancestral sequence reconstruction allows us to deduce the amino acid sequences of ancestral proteins. Here, we present the structural and functional characteristics of an ancestral protein, AncMH, reconstructed to be the last common ancestor of hemoglobins and myoglobins. Our findings reveal that AncMH harbors heme and that the heme binds oxygen. Furthermore, we demonstrate that the ferrous heme in AncMH is pentacoordinated, similar to that of human adult hemoglobin and horse myoglobin. A detailed comparison of the heme pocket structure indicates that the heme pocket in AncMH is more similar to that of hemoglobin than that of myoglobin. However, the autoxidation of AncMH is faster than that of both hemoglobin and myoglobin. Collectively, our results suggest that ancestral proteins of hemoglobins and myoglobins evolved in steps, including the hexa- to pentacoordination transition, followed by stabilization of the oxygen-bound form.
Topics: Adult; Humans; Animals; Horses; Globins; Heme; Myoglobin; Amino Acid Sequence; Oxygen
PubMed: 37647623
DOI: 10.1021/acs.biochem.3c00203 -
Pulse Duration Effects on Solution-Phase Protein Desorption in Laser Electrospray Mass Spectrometry.Journal of the American Society For... Apr 2023The effect of laser pulse duration on the ablation of aqueous myoglobin is investigated using laser electrospray mass spectrometry (LEMS). Pulse durations of 55...
The effect of laser pulse duration on the ablation of aqueous myoglobin is investigated using laser electrospray mass spectrometry (LEMS). Pulse durations of 55 femtoseconds (fs), 56 piscoseconds (ps), and 10 nanoseconds (ns) were used to ablate aqueous myoglobin from stainless-steel and quartz substrates. The integrated signal intensity of myoglobin increases with decreasing pulse duration for both substrates. Laser-induced thermal effects are assessed by the relative amount of solvent adduction and number of phosphate moieties adducted to myoglobin by each laser pulse duration. The mass spectra for 55 fs vaporization shows myoglobin with appreciable solvent and phosphate adduction and baseline elevation. The mass spectra for 10 ns ablation have minimal adduction and limited baseline elevation. Heat-induced conformation changes in myoglobin were used to measure the amount of thermal energy deposited by each laser pulse duration. Ablation using the 55 fs pulse revealed the highest ratio of unfolded to folded myoglobin in comparison to the 56 ps and 10 ns measurements due to increased droplet lifetime and consequent interaction with the acid in the electrospray solvent. Collisional activation and heated capillary temperature were employed to reduce the droplet lifetime and demonstrate that fs ablation preserves approximately 2 times more myoglobin folded conformation in comparison to ps and ns pulses.
Topics: Myoglobin; Spectrometry, Mass, Electrospray Ionization; Muramidase; Cytochromes c; Lasers; Solvents; Water
PubMed: 36947866
DOI: 10.1021/jasms.2c00362 -
Journal of Biological Inorganic... May 2020Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to...
Myoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., k) is (6.8 ± 0.7) × 10 M s and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., k) is 3.1 ± 0.3 s. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., k/k = (4.6 ± 0.7) × 10 M) is virtually the same as that directly measured (i.e., K = (3.8 ± 0.5) × 10 M). In the absence of NO, Mb(III) catalyzes the conversion of peroxynitrite to NO, the value of the second-order rate constant (i.e., k) being (1.9 ± 0.2) × 10 M s. However, in the presence of NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (k* = (2.8 ± 0.3) × 10 M s). These data expand the multiple roles of NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO.
Topics: Animals; Catalysis; Ferric Compounds; Free Radical Scavengers; Male; Molecular Structure; Myoglobin; Nitro Compounds; Peroxynitrous Acid; Whales
PubMed: 32172452
DOI: 10.1007/s00775-020-01767-2 -
Food Chemistry Aug 2024(-)-Epigallocatechin-3-gallate (EGCG) is remarkably efficacious in inhibiting the browning of red meat. We therefore propose a hypothesis that EGCG forms complexes with...
(-)-Epigallocatechin-3-gallate (EGCG) is remarkably efficacious in inhibiting the browning of red meat. We therefore propose a hypothesis that EGCG forms complexes with myoglobin, thereby stabilizing its structure and thus preventing browning. This study investigated the interaction mechanism between EGCG and myoglobin. EGCG induced static quenching of myoglobin. Noncovalent forces, including hydrogen bonds and van der Waals, primarily governing the interactions between myoglobin and EGCG. The interactions primarily disrupted myoglobin's secondary structure, thus significantly reducing surface hydrophobicity by 53% (P < 0.05). The modification augmented the solubility and thermal stability of myoglobin. The radius of gyration (Rg) value fluctuated between 1.47 and 1.54 nm, and the hydroxyl groups in EGCG formed an average of 2.93 hydrogen bonds with myoglobin. Our findings elucidated the formation of stable myoglobin-EGCG complexes and the myoglobin-EGCG interaction, thus confirming our initial hypothesis.
Topics: Myoglobin; Catechin; Hydrophobic and Hydrophilic Interactions; Hydrogen Bonding; Animals; Protein Binding
PubMed: 38608400
DOI: 10.1016/j.foodchem.2024.139208 -
Journal of Inorganic Biochemistry Aug 2024Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been... (Review)
Review
Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.
Topics: Neuroglobin; Myoglobin; Globins; Nerve Tissue Proteins; Humans; Animals; Heme; Binding Sites; Metalloproteins; Protein Engineering
PubMed: 38759262
DOI: 10.1016/j.jinorgbio.2024.112595 -
Meat Science Dec 2023Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry...
Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry beef, often referred to as 'dark-cutting'. However, the relationship between atypical dark (AT) beef, postmortem energy metabolism and underlying tissue characteristics remains somewhat unclear. Cattle harvested in the US and Canada representing normal (pH < 5.6), AT dark (pH 5.6-5.8) and dark cutting (DC; pH > 5.8) beef were analyzed for tissue characteristics related to energy metabolism. Results show AT dark beef is more oxidative but similar to normal beef in glycolytic potential and nucleotide abundance. Mitochondria DNA content (P < 0.05, Canada; P < 0.005, US) and oxidative enzymes for DC and AT dark beef were greater (P < 0.01; Canada and US) compared to normal beef. Myoglobin tracked (P < 0.01) with color classification. These findings show both DC and AT beef are inherently more oxidative and raise the possibility that more oxidative muscle may be more prone to develop dark beef.
Topics: Cattle; Animals; Muscle, Skeletal; Color; Myoglobin; Glycogen; Glycolysis; Hydrogen-Ion Concentration; Red Meat
PubMed: 37778129
DOI: 10.1016/j.meatsci.2023.109344 -
International Journal of Biological... Dec 2021Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other...
Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other hand, Vitamin B12 is a vital nutrient that helps synthesize red blood cells, DNA, and proteins. To understand the ability of vitamin B12 to bind to the excess of myoglobin produced in the body under certain conditions (muscle injuries, severe trauma, etc.), it is essential to dig into the interaction between them. Therefore, the present study reports the binding interaction of vitamin B12 and myoglobin employing different spectroscopic and computational methods. The myoglobin's intrinsic fluorescence is quenched by vitamin B12 via static nature as observed from steady-state as well as time-resolved fluorescence measurements. The microenvironment of myoglobin's tryptophan residue gets affected, but there is no change observed in its α-helical content by vitamin B12 as seen from synchronous fluorescence and circular dichroism measurements. The probable binding of vitamin B12 on myoglobin was elucidated through molecular docking, and the interaction stability was studied by molecular dynamics simulation. The determination of vitamin B12's affinity to myoglobin and its effect on the conformational transitions of myoglobin might afford valuable insight for clinical pharmacology.
Topics: Binding Sites; Circular Dichroism; Humans; Hydrogen Bonding; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Myoglobin; Protein Binding; Spectrum Analysis; Vitamin B 12
PubMed: 34653439
DOI: 10.1016/j.ijbiomac.2021.10.030 -
Scientific Reports Jun 2021The presence of deoxygenated hemoglobin (Hb) results in a drop in T2 and T2* in magnetic resonance imaging (MRI), known as the blood oxygenation level-dependent...
The presence of deoxygenated hemoglobin (Hb) results in a drop in T2 and T2* in magnetic resonance imaging (MRI), known as the blood oxygenation level-dependent (BOLD-)effect. The purpose of this study was to investigate if deoxygenated myoglobin (Mb) exerts a BOLD-like effect. Equine Met-Mb powder was dissolved and converted to oxygenated Mb. T1, T2, T2*-maps and BOLD-bSSFP images at 3Tesla were used to scan 22 Mb samples and 12 Hb samples at room air, deoxygenation, reoxygenation and after chemical reduction. In Mb, T2 and T2* mapping showed a significant decrease after deoxygenation (- 25% and - 12%, p < 0.01), increase after subsequent reoxygenation (+ 17% and 0% vs. room air, p < 0.01), and finally a decrease in T2 after chemical reduction (- 28%, p < 0.01). An opposite trend was observed with T1 for each stage, while chemical reduction reduced BOLD-bSSFP signal (- 3%, p < 0.01). Similar deflections were seen at oxygenation changes in Hb. The T1 changes suggests that the oxygen content has been changed in the specimen. The shortening of transverse relaxation times in T2 and T2*-mapping after deoxygenation in Mb specimens are highly indicative of a BOLD-like effect.
Topics: Animals; Hemoglobins; Horses; Humans; Magnetic Resonance Imaging; Myoglobin; Oxygen
PubMed: 34075096
DOI: 10.1038/s41598-021-90908-x -
Nitric Oxide : Biology and Chemistry Sep 2019The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO) and nitrite (NO) has gained increasing attention due to the potential beneficial effects of...
The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO) and nitrite (NO) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle. We observed a modest but significant decrease of nitrate level in skeletal muscle of myoglobin deficient mice compared to littermate control mice (17.3 vs 12.8 nmol/g). In contrast, a NOS inhibitor, L-NAME or a low nitrite/nitrate diet treatment led to more pronounced decreases of nitrate levels in the skeletal muscle of both control and myoglobin deficient mice. Nitrite levels in the skeletal muscle of both types of mice were similar (0.48 vs 0.42 nmol/g). We also analyzed the expression of several proteins that are closely related to NO metabolism to examine the mechanism by which nitrate and nitrite levels are preserved in the absence of myoglobin. Western blot analyses suggest that the protein levels of xanthine oxidoreductase and sialin, a nitrate transporter, both increased in the skeletal muscle of myoglobin deficient mice. These results are compatible with our previously reported model of nitrate production in muscle and suggest that myoglobin deficiency activates compensatory mechanisms to sustain NO homeostasis.
Topics: Animals; Homeostasis; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Myoglobin; Nitric Oxide
PubMed: 31173908
DOI: 10.1016/j.niox.2019.06.001 -
Molecular Pharmaceutics Aug 2023The effects of atomic layer (ALC) coating on physical properties and storage stability were examined in solid powders containing myoglobin, a model protein. Powders...
The effects of atomic layer (ALC) coating on physical properties and storage stability were examined in solid powders containing myoglobin, a model protein. Powders containing myoglobin and mannitol (1:1 w/w) were prepared by lyophilization or spray drying and subjected to aluminum oxide or silicon oxide ALC coating. Uncoated samples of these powders as well as coated and uncoated samples of myoglobin as received served as controls. After preparation (), samples were analyzed for moisture content, reconstitution time, myoglobin secondary structure, crystallinity, and protein aggregate content. Samples were stored for 3 months () under controlled conditions (53% RH, 40 °C) in both open and closed vials and then analyzed as above. At , the recovery of soluble native (i.e., monomeric) protein depended on formulation, coating type, and drying method and was up to 2-fold greater in coated samples than in uncoated controls. Promisingly, some samples with high recovery also showed low soluble aggregate content (<10%) at and low total monomer loss; the latter was correlated to sample moisture content. Overall, the results demonstrate that ALC coatings can stabilize solid protein formulations during storage, providing benefits over uncoated controls.
Topics: Myoglobin; Powders; Freeze Drying; Protein Structure, Secondary; Drug Stability
PubMed: 37466053
DOI: 10.1021/acs.molpharmaceut.3c00229