Protein Myoglobin Research Articles

Ultrafast Structural Fluctuations of Myoglobin-Bound Thiocyanate and Selenocyanate Ions Measured with Two-Dimensional Infrared Photon Echo Spectroscopy.

Structural dynamics within the distal cavity of myoglobin protein is investigated using 2D-IR and IR pump-probe spectroscopy of the N≡C stretch modes of heme-bound thiocyanate and selenocyanate ions. Although myoglobin-bound thiocyanate group shows a doublet in its IR absorption spectrum, no cross peaks originating from chemical exchange between the two components are observed in the time-resolved 2D IR spectra within the experimental time window. Frequency-frequency correlation functions of the two studied anionic ligands are obtained by means of a few different analysis approaches; these functions were then used to elucidate the differences in structural fluctuation around ligand, ligand-protein interactions, and the degree of structural heterogeneity within the hydrophobic pocket of these myoglobin complexes.

Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation.

The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity.

Improvement of Endurance Based on Muscle Fiber-Type Composition by Treatment with Dietary Apple Polyphenols in Rats.

A recent study demonstrated a positive effect of apple polyphenol (APP) intake on muscle endurance of young-adult animals. While an enhancement of lipid metabolism may be responsible, in part, for the improvement, the contributing mechanisms still need clarification. Here we show that an 8-week intake of 5% (w/w) APP in the diet, up-regulates two features related to fiber type: the ratio of myosin heavy chain (MyHC) type IIx/IIb and myoglobin protein expression in plantaris muscle of 9-week-old male Fischer F344 rats compared to pair-fed controls (P < 0.05). Results were demonstrated by our SDS-PAGE system specialized for MyHC isoform separation and western blotting of whole muscles. Animal-growth profiles (food intake, body-weight gain, and internal-organ weights) did not differ between the control and 5% APP-fed animals (n = 9/group). Findings may account for the increase in fatigue resistance of lower hind limb muscles, as evidenced by a slower decline in the maximum isometric planter-flexion torque generated by a 100-s train of electrical stimulation of the tibial nerve. Additionally, the fatigue resistance was lower after 8 weeks of a 0.5% APP diet than after 5% APP, supporting an APP-dose dependency of the shift in fiber-type composition. Therefore, the present study highlights a promising contribution of dietary APP intake to increasing endurance based on fiber-type composition in rat muscle. Results may help in developing a novel strategy for application in animal sciences, and human sports and age-related health sciences.

Synthesis of coumarin-appended cyclophanes and evaluation of their complexation with myoglobin

Coumarin-appended cyclophanes bearing positively or negatively charged side chains were synthesized as a water-soluble host (1a or 1b, respectively). Host 1a and 1b showed fluorescence bands with fluorescence maxima at 404 nm originated from coumarin moiety. As a host for guest molecules by using macrocyclic cavity, cationic host 1a binds anionic guests such as 6-p-toluidinonaphthalene-2-sulfonate (TNS), 6-anilinonaphthalene-2-sulfonate (2,6-ANS), and 8-anilinonaphthalene-1-sulfonate (1,8-ANS) more strongly than anionic host 1b, reflecting intermolecular electrostatic interactions. In addition, both host 1a and 1b showed protein surface recognition and fluorescence response toward myoglobin, a small and globular protein. The fluorescence intensity originating from the hosts decreased upon the addition of myoglobin, reflecting the formation of 1a- and 1b-myoglobin complexes. On the other hand, such fluorescence response of 1a and 1b was almost negligible for other proteins such as egg white albumin, bovine serum albumin, human albumin, concanavaline A, fibrinogen, γ-globlin, peanut agglutinin, trypsin, and lysozyme.

Composition dependent multiple structural transformations of myoglobin in aqueous ethanol solution: a combined experimental and theoretical study.

Experimental studies (circular dichroism and ultra-violet (UV) absorption spectra) and large scale atomistic molecular dynamics simulations (accompanied by order parameter analyses) are combined to establish a number of remarkable (and unforeseen) structural transformations of protein myoglobin in aqueous ethanol mixture at various ethanol concentrations. The following results are particularly striking. (1) Two well-defined structural regimes, one at xEtOH ∼ 0.05 and the other at xEtOH ∼ 0.25, characterized by formation of distinct partially folded conformations and separated by a unique partially unfolded intermediate state at xEtOH ∼ 0.15, are identified. (2) Existence of non-monotonic composition dependence of (i) radius of gyration, (ii) long range contact order, (iii) residue specific solvent accessible surface area of tryptophan, and (iv) circular dichroism spectra and UV-absorption peaks are observed. Interestingly at xEtOH ∼ 0.15, time averaged value of the contact order parameter of the protein reaches a minimum, implying that this conformational state can be identified as a molten globule state. Multiple structural transformations well known in water-ethanol binary mixture appear to have considerably stronger effects on conformation and dynamics of the protein. We compare the present results with studies in water-dimethyl sulfoxide mixture where also distinct structural transformations are observed along with variation of co-solvent composition.

Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state.

‘Cool’ adaptations to cold environments: globins in Notothenioidei (Actynopterygii, Perciformes)

Notothenioidei, the taxonomic group of teleosts that dominates the Southern Ocean and dwell in the Ross Sea at large, provide an example of marine species that underwent unique adaptations to life at low temperatures and high oxygen concentrations, resulting in morphological, physiological, genomic, and biochemical peculiarities in comparison with warm-water fish. Global Warming raises concerns over the fate of these stenothermal fish, as their adaptation has been accompanied by irreversible genomic losses, which suggest a poor genetic potential to adapt to warmer climates. Specifically, this review focuses on adaptation of proteins belonging to the globin superfamily, which include the respiratory proteins hemoglobin and myoglobin and the non-respiratory proteins neuroglobin and cytoglobin. Here, we describe their molecular adaptations to cold temperatures in the framework of the physiology of oxygen transport and management of oxidative stress in fish species largely populating the Ross Sea.

Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)‐Substituted Polyoxometalates

SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site.

Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)‐Substituted Polyoxometalates

AbstractSDS‐PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)‐substituted Lindqvist‐, Keggin‐, and Wells–Dawson‐type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at AspX peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein‐hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site.

Celebrating macromolecular crystallography: A personal perspective

The twentieth century has seen an enormous advance in the knowledge of the atomic structures that surround us. The discovery of the first crystal structures of simple inorganic salts by the Braggs in 1914, using the diffraction of X-rays by crystals, provided the critical elements to unveil the atomic structure of matter. Subsequent developments in the field leading to macromolecular crystallography are presented with a personal perspective, related to the cultural milieu of Spain in the late 1950’s. The journey of discovery of the author, as he developed professionally, is interwoven with the expansion of macromolecular crystallography from the first proteins (myoglobin, hemoglobin) to the ‘coming of age’ of the field in 1971 and the discoveries that followed, culminating in the determination of the structure of the ribosomes at the turn of the century. A perspective is presented exploring the future of the field and also a reflection about the future generations of Spanish scientists.

Protein Attachment on Nanodiamonds.

A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry.

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N(2) bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N(2) bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N(2) sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, "semi-unfolded" ↔ "native" ↔ "globally unfolded" → "aggregated". This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS.

Get up and Learn – Active Learning of Hemoglobin Function in the Biochemistry Classroom

Constructivist learning theory, as formalized by Piaget, suggests that learners construct new knowledge from their experiences and is best facilitated with pedagogic approaches that promote learning by doing. A traditional stumbling block in undergraduate biochemistry is the structure‐function relationship in proteins. Students often first encounter this relationship in the study of the oxygen‐binding proteins hemoglobin and myoglobin. While the molecular structures and physiological roles of these proteins are known in detail, the interplay of additional components (e.g. carbonic anhydrase, O2/CO2 levels, 2,3‐BPG) can make this dance in the circulatory system a challenge for students to master conceptually. In this study, an active learning approach through a hands‐on game was used to facilitate student mastery, whereby students become the molecules responsible for oxygen transport in the circulatory system. By students having a greater understanding of how this system works properly in a healthy state, they can better understand aspects that result in disease (e.g. Sickle‐cell anemia and hemoglobin variants) or predict outcomes arising from changes in environmental conditions (e.g. high altitude or hyperventilation). The objective of this study is to determine if there is any difference between perceived and actual success in learning outcomes as students partake in this alternative pedagogical approach. The working hypothesis is that utilization of constructivist pedagogies, such as an active learning game, will result in higher perceived and actual student success. Data is being collected during the Fall 2015 semester to evaluate this hypothesis.

Denaturation properties and folding transition states of leghemoglobin and other heme proteins.

This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.

Top-down MALDI-in-source decay-FTICR mass spectrometry of isotopically resolved proteins.

An accurate mass measurement of a known protein provides information on potential amino acid deletions and post-translational modifications. Although this field is dominated by strategies based on electrospray ionization, mass spectrometry (MS) methods using matrix-assisted laser desorption/ionization (MALDI) have the advantage of yielding predominantly singly charged precursor ions, thus avoiding peak overlap from different charge states of multiple species. Such MALDI-MS methods require mass measurement at ultrahigh resolution, which is provided by Fourier transform ion cyclotron resonance (FTICR) mass analyzers. Recently, using a MALDI-FTICR-MS platform equipped with a 15 T magnet, we reported on the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ∼15 kDa and identified new proteoforms from an accurate measurement of mass distances. In the current study, we have used this FTICR system after an upgrade with a novel dynamically harmonized ICR cell, i.e., ParaCell, for mapping isotopically resolved intact proteins up to about 17 kDa and performed top-down MALDI in-source decay (ISD) analysis. Standard proteins myoglobin (m/z-value 16,950) and ribonuclease B (m/z-value 14,900) were measured with resolving powers of 62,000 and 61,000, respectively. Furthermore, it will be shown that (singly charged) MALDI-ISD fragment ions can be measured at isotopic resolution up to m/z-value 12,000 (e.g., resolving power 39,000 at m/z-value 12,000) providing more reliable identifications. Moreover, examples are presented of pseudo-MS(3) experiments on ISD fragment ions from RNase B by collisional-induced dissociation (CID).

The influence of estradiol on muscle damage and leg strength after intense eccentric exercise.

To examine the influence of estradiol on muscle damage and leg strength after intense eccentric exercise. Eight men (MEN), eight normally menstruating women (WomenNM), and eight women using oral contraceptives (WomenOC) participated in this study. Subjects performed 240 maximal-effort bilateral eccentric contractions of the quadriceps muscle groups designed to elicit exercise-induced muscle damage (EiMD). Serum creatine kinase (CK), myoglobin (Mb), and fatty acid-binding protein (FABP) concentrations were measured before (pre-) EiMD, as well as 0, 6, 24, and 48 h post-EiMD. Peak isometric quadriceps torque (i.e., leg strength) was measured pre-EiMD, as well as 24 and 48 h post-EiMD. The increases in CK, Mb, and FABP concentrations from pre- to post-EiMD were greater in MEN (10-fold, 15-fold, and fourfold, respectively) and WomenOC (sevenfold, 11-fold, and ninefold) compared with WomenNM (five-, six-, and threefold; p < 0.05). The decline in leg strength was about 10 % pre- to 24 h post-EiMD in all groups and decreased a further 10-15 % by 48 h post-EiMD in the MEN and WomenOC only. Our findings suggest an important role of estradiol in blunting the muscle damage response to intense eccentric exercise and preserving muscle function after EiMD.

Hofmeister ion effects on the solvation and thermal stability of model proteins lysozyme and myoglobin

Circular dichroism (CD) and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopies were used to study how sucrose and salts, NaCl, NaClO4, Na2SO4, CaCl2, MgCl2, MgSO4, and NH4Cl alter lysozyme and myoglobin solvation and thermal stability. Both proteins follow the anionic Hofmeister series in which chaotropes (Cl−, ClO4−) decrease thermal stability, and kosmotropes (SO42−) increase thermal stability. CD spectra showed no significant secondary structural differences at 25 °C that correlate with changes in thermal stability. However, infrared spectra of lysozyme and myoglobin show a downshift in the Amide I′ (backbone CO) vibration and increased 1H/2H exchange in the presence of weakly hydrated, chaotropic anions (Cl−, ClO4−), both of which suggest increased protein solvation in the presence of chaotropes and correlate with decreased protein thermal stability. The presence of strongly hydrated, kosmotropic anions (SO42−) or sucrose prevent the downshift of the Amide I′ vibration in lysozyme and myoglobin and result in decreased 1H/2H exchange, suggesting decreased protein solvation in the presence of kosmotropes, that correlates with increased protein stability. The data show a strong correlation between the level of deuterium exchange and the relative thermal stability in the presence of kosmotropes or chaotropes that does not correlate with secondary structural changes and suggests that Hofmeister ions alter protein solvation. These results also show that a combination of CD and infrared spectroscopy is useful in studying how Hofmeister ions and co-solvents influence protein solvation and thermal stability.

Changesin Myoglobin of Big Eye Tuna During Chilling Storage

Big eye tuna (Thunnus obesus) is one of the species of tuna which is have some value added such as have potential to improve animal protein sources, have high economic values as well as an export commodity.Mishandling and misapplication of high temparatures on the tuna handling at the tropics and sub tropics climate was significantly decreasing the value of myoglobin and affecting the solubility of protein. Myoglobin is a globular protein that have small molecular weight size and it was an important factor for determining the quality of meat and influencing factors of purchasing power by the consumer. The purpose of this experiments is to determining the changes of myoglobin content and the water soluble proteins content at some parts of big eye tuna in 9 days chilling temperatures. The portion which is analized was the ventral area, dorsal area and tail area. Myoglobin content in all portion above, both light and dark meat was analized. The results shows the decreased value of myoglobin content from first handling (day zero) until day ninth (days 9th) experiment. Each myoglobin contentfrom white meat at at ventral, dorsal and tail meat was decreased from 121.68 mg/100 into 41.35 mg/100, 148.2 mg/100g into 52.01 mg/100g, 105.16 mg/100g into 31.34 gr/100gram, after day ninth. The myoglobin content from dark meat at ventral, dorsal and tail meat, was decreased, too ; from 418.64 mg/100 gr into 121.01 mg/100 g, 446.21 mg/100 g into 58.34 mg/100 r and 145.65 mg/100 gr into 87.98 mg/100g after day ninth.Water soluble protein was derived into protein bands with molecular weight 15,4 kDa and 14 kDa. Its suspected as the myoglobin protein. The molecular weight difference was caused from degradation of protein during the storage.&lt;br /&gt;Keywords: Big eye tuna, meat, storage, myoglobin

In Silico Characterization and Comparative Analysis of Allergenicity of Allergic Proteins from Different Food Sources

Allergy is a steadily increasing health problem for all age groups. In general, it’s recommended that 10-35% of our daily calories come from protein. A complete protein source is one that provides all of the essential amino acids called high quality proteins. Although animal-based foods; for example, meat, poultry, fish, milk, eggs, are considered complete protein sources these are the common sources of causing allergenicity. In case of meat Bos taurus, prawn Penaeus monodon and egg Gallus gallus are found to be the most responsible for triggering allergenicity. The current study has disclosed the best alternative sources for meat, prawn and egg through in silico characterization and comparative analysis of allergic proteins (Myoglobin Ovomucoid, Lysozyme, Ovalbumin, Ovotransferrin, Tropomyosin) with other common sources of meat (Capra hircus, Ovis aries, Gallus gallus, Sus scrofa), prawn (Fenneropenaeus merguiensis, Macrobrachium rosenbergii, Metapenaeus ensis, Pandalus borealis) and egg (Anas platyrhynchos). Analyzing the results we found that Gallus gallus, Macrobrachium rosenbergii, Anas platyrhynchos would be the safe source for meat, prawn and egg respectively.