Showing papers on "Myoglobin published in 2015"

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

Abstract: 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.

Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser

Abstract: Light absorption can trigger biologically relevant protein conformational changes The light-induced structural rearrangement at the level of a photoexcited chromophore is known to occur in the femtosecond timescale and is expected to propagate through the protein as a quake-like intramolecular motion Here we report direct experimental evidence of such 'proteinquake' observed in myoglobin through femtosecond X-ray solution scattering measurements performed at the Linac Coherent Light Source X-ray free-electron laser An ultrafast increase of myoglobin radius of gyration occurs within 1 picosecond and is followed by a delayed protein expansion As the system approaches equilibrium it undergoes damped oscillations with a ~36-picosecond time period Our results unambiguously show how initially localized chemical changes can propagate at the level of the global protein conformation in the picosecond timescale

Intermolecular carbene S-H insertion catalysed by engineered myoglobin-based catalysts†.

Abstract: The first example of a biocatalytic strategy for the synthesis of thioethers via an intermolecular carbene S–H insertion reaction is reported. Engineered variants of sperm whale myoglobin were found to efficiently catalyze this C–S bond forming transformation across a diverse set of aryl and alkyl mercaptan substrates and α-diazoester carbene donors, providing high conversions (60–99%) and high numbers of catalytic turnovers (1100–5400). Furthermore, the enantioselectivity of these biocatalysts could be tuned through mutation of amino acid residues within the distal pocket of the hemoprotein, leading to myoglobin variants capable of supporting asymmetric S–H insertions with up to 49% ee. Rearrangement experiments support a mechanism involving the formation of a sulfonium ylide generated upon attack of the thiol substrate to a heme-bound carbene intermediate.

Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions.

Abstract: Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly.

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

Abstract: 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.

Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected.

Abstract: The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 A, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity.

Visual detection of myoglobin via G-quadruplex DNAzyme functionalized gold nanoparticles-based colorimetric biosensor

Abstract: Since myoglobin plays a major role in the diagnosis of acute myocardial infarction (AMI), monitoring of myoglobin in point-of-care is fundamental. Here, a novel colorimetric assay for myoglobin detection was developed based on hemin/G-quadruplex DNAzyme functionalized gold nanoparticles (AuNPs). In the presence of myoglobin, the anti-myoglobin antibody, which was modified on the surface of polystyrene microplate, could first capture the target myoglobin. Then the captured target could further bind to DNA1 probe which contained the aptamer sequence through aptamers/myoglobin interaction. Next, as the DNA2 probe modified AuNPs were introduced, DNA2 probe modified AuNPs could hybridize with the captured DNA1 probe. Subsequently, DNA2 probe which was modified on the AuNPs could fold into a G-quadruplex structure and bind to hemin, and then catalyze the oxidation of colorless ABTS 2− to green ABTS + by H 2 O 2 . Consequently, the relationship between the concentration of myoglobin and the absorbance was established. Due to AuNPs amplification, the myoglobin concentration as low as 2.5 nM could be detected, which was lower than clinical cutoff for myoglobin in healthy patients. This assay also showed high selectivity for myoglobin and was used for the detection of myoglobin in the human serum samples. This work may provide a simple but effective tool for early diagnosis of AMI in the world, especially in developing countries.

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy

Abstract: We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin- and a peroxidase-like heme-protein.

Abstract: Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc.

Protein Attachment on Nanodiamonds

Abstract: 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.

An Electrochemical Biosensor Based on a Myoglobin-specific Binding Peptide for Early Diagnosis of Acute Myocardial Infarction

Abstract: In this study, a simple, highly sensitive electrochemical biosensor for myoglobin was developed using a myoglobin-specific binding peptide as a sensing probe. A peptide (Myo-3R7, CPSTLGASC, 838 Da) identified by phage display and that specifically binds to myoglobin was covalently immobilized on a gold electrode functionalized via a dithiobis(succinimidyl propionate) (DSP) self-assembled monolayer (SAM). The peptide immobilization was confirmed with fluorescence microarray scanning and cyclic voltammetry (CV). The electrochemical performance of the biosensor with respect to myoglobin was characterized by CV and differential pulse voltammetry (DPV) using Fe(CN)6(3-)/Fe(CN)6(4-) as a redox probe. We successfully detected myoglobin in a broad working range of 17.8 to 1780 ng mL(-1) with a correlation coefficient (R(2)) of 0.998. The estimated limit of detection (LOD) was fairly low, 9.8 ng mL(-1) in 30 min. The electrochemical biosensor based on a myoglobin-specific binding peptide offers sensitivity, selectivity, and rapidity, making it an attractive tool for the early detection of cardiac infarction.

In vivo photoacoustic tomography of myoglobin oxygen saturation

Abstract: Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen (O_2) concentration in response to hypoxia or elevated muscle activities. We present a method that uses photoacoustic computed tomography to measure the distribution of myoglobin in tissue and the oxygen saturation of myoglobin (sO_2-Mb). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the sO_2 –Mb change in the backbone muscle in vivo.

Interaction of Myoglobin with Cationic Gemini Surfactants in Phosphate Buffer at pH 7.4

Abstract: The interactions between myoglobin, a monomeric water soluble heme protein, and cationic gemini surfactants 14-3-14, 14-4-14, 14-5-14 have been studied in phosphate buffer at pH 7.4 using different techniques such as surface tension, UV–visible spectroscopy, fluorescence spectroscopy and circular dichroism. Myoglobin is a surface active protein and it is bound with surfactant molecules. The spin states of the heme group of myoglobin change depending on the concentration of surfactant. The unfolding process of protein in the presence of surfactant is observed from fluorescence spectra. With increasing surfactant concentration, α-helicity of myoglobin decreases with the appearance of β-sheet and random coil. The extent of interaction of myoglobin with gemini surfactant increases with increasing spacer length.

Changes in whole tissue heme concentration dissociates muscle deoxygenation from muscle oxygen extraction during passive head-up tilt

Abstract: Skeletal muscle deoxygenated hemoglobin and myoglobin concentration ([HHb]), assessed by near-infrared spectroscopy (NIRS), is commonly used as a surrogate of regional O2 extraction (reflecting the...

Generation of New Artificial Metalloproteins by Cofactor Modification of Native Hemoproteins

Abstract: Heme can be removed from a number of native hemoproteins, thus forming corresponding apoproteins, each of which provides a site for binding of a metal complex. In one example, myoglobin, an O2 storage protein, can be reconstituted with iron porphycene to dramatically enhance the O2 affinity. Although it is known that myoglobin has poor enzymatic activity, the insertion of iron corrole or iron porphycene into apomyoglobin increases its H2O2-dependent peroxidase/peroxygenase activities. Furthermore, reconstitution with manganese porphycene promotes hydroxylation of an inert CH bond. It is also of interest to insert a non-porphyrinoid complex into an apoprotein. A cavity of apocytochrome c has been found to bind a diiron carbonyl complex, serving as a functional model of diiron hydrogenase. Aponitrobindin has a rigid β-barrel structure that provides an excellent cavity for covalently anchoring a metal complex. A rhodium complex embedded in the cavity of genetically modified nitrobindin has been found to promote stereoselective polymerization of phenylacetylene.

Heme iron in meat as the main source of iron in the human diet

Abstract: Iron is a trace element involved in many cardinal metabolic processes of almost all living organisms It is well known that iron participates in oxygen transport as well as it is a cofactor in many fundamental enzymatic and nonenzymatic processes Accordingly, disturbances of iron homeostasis can cause serious clinical consequences In humans, dietary iron can enter the body in two main forms: heme and nonheme The former is a component of many hemoproteins (including myoglobin, hemoglobin, cytochromes b and c) and is easily absorbed in the duodenal enterocytes Red meat is an excellent source of heme iron, while the less bioavailable nonheme form is found in large amounts in milk products and vegetables For this reason, consumers of meat have a better iron status than vegetarians and vegans Heme iron found in muscle protein should be supplied to humans to prevent iron deficiency, which can lead to anemia It is easily absorbed and its main source is red meat In addition, heme iron, which is mainly found in myoglobin in meat, contributes to the desirable bright red color and to the most undesirable brown color of meat Both heme and nonheme iron are catalysts of lipid oxidation in meat This process lowers the nutritive value through oxidation of polyunsaturated fatty acids, which produces an undesirable flavor and aroma The aim of this paper was to discuss the role of heme iron in the human diet

Evaluation of Medicine Effects on the Interaction of Myoglobin and Its Aptamer or Antibody Using Atomic Force Microscopy

Abstract: The effects of medicine on the biomolecular interaction have been given increasing attention in biochemistry and affinity-based analytics since the environment in vivo is complex especially for the patients. Herein, myoglobin, a biomarker of acute myocardial infarction, was used as a model, and the medicine effects on the interactions of myoglobin/aptamer and myoglobin/antibody were systematically investigated using atomic force microscopy (AFM) for the first time. The results showed that the average binding force and the binding probability of myoglobin/aptamer almost remained unchanged after myoglobin-modified gold substrate was incubated with promazine, amoxicillin, aspirin, and sodium penicillin, respectively. These parameters were changed for myoglobin/antibody after the myoglobin-modified gold substrate was treated with these medicines. For promazine and amoxicillin, they resulted in the change of binding force distribution of myoglobin/antibody (i.e., from unimodal distribution to bimodal distribut...

Molecular mechanism of myoglobin autoxidation: insights from computer simulations.

Abstract: Myoglobin (Mb) and hemoglobin have the biological ability to carry/store oxygen (O2), a property which requires its heme iron atom to be in the ferrous--Fe(II)--state. However, the thermodynamically stable state in the presence of O2 is Fe(III) and thus the oxidation rate of a globin is a critical parameter related to its function. Mb has been extensively studied and many mutants have been characterized regarding its oxygen mediated oxidation (i.e., autoxidation) rates. Site directed mutants in residues 29 (B10), which shapes the distal cavity, and 64 (E7), the well-known histidine gate, have been shown to display a wide range of autoxidation rate constants. In this work, we have thoroughly studied the mechanism underlying the autoxidation process by means of state-of-the-art computer simulation methodologies, using Mb and site directed mutants as benchmark cases. Our results explain the observed autoxidation rate tendencies in different variants of Mb, L29F < wt < L29A = H64Q < H64F < H64A, and shed light on several aspects of the reaction at the atomic level. First, water access to the distal pocket is a key event and the observed acid catalysis relies on HisE7 protonation and opening of the His gate to allow water access, rather than protonation of the oxy heme itself. Our results also suggest that the basic mechanism, i.e., superoxide displacement by hydroxide anion, is energetically more feasible. Finally, we confirmed that distal hydrogen bonds protect the oxy complex from autoxidation.

How a novel tyrosine–heme cross-link fine-tunes the structure and functions of heme proteins: a direct comparitive study of L29H/F43Y myoglobin

Abstract: A heme–protein cross-link is a key post-translational modification (PTM) of heme proteins. Meanwhile, the structural and functional consequences of heme-protein cross-links are not fully understood, due to limited studies on a direct comparison of the same protein with and without the cross-link. A Tyr–heme cross-link with a C–O bond is a newly discovered PTM of heme proteins, and is spontaneously formed in F43Y myoglobin (Mb) between the Tyr hydroxyl group and the heme 4-vinyl group in vivo. In this study, we found that with an additional distal His29 introduced in the heme pocket, the double mutant L29H/F43Y Mb can form two distinct forms under different protein purification conditions, with and without a novel Tyr–heme cross-link. By solving the X-ray structures of both forms of L29H/F43Y Mb and performing spectroscopic studies, we made a direct structural and functional comparison in the same protein scaffold. It revealed that the Tyr–heme cross-link regulates the heme distal hydrogen-bonding network, and fine-tunes not only the spectroscopic and ligand binding properties, but also the protein reactivity. Moreover, the formation of the Tyr–heme cross-link in the double mutant L29H/F43Y Mb was investigated in vitro. This study addressed the key issue of how Tyr–heme cross-link fine-tunes the structure and functions of the heme protein, and provided a plausible mechanism for the formation of the newly discovered Tyr–heme cross-link.

Resonance Raman detection of the myoglobin nitrito heme Fe–O–NO/2-nitrovinyl species: implications for helix E-helix F interactions

Abstract: The description of biological activity in heme proteins responsible for activating small molecules requires identification of ligand movement into the metal and non-metal binding sites. Mechanisms of nitrite reductase activity in globins are difficult to verify without the structures of the bound ligand, but we now have such information from resonance Raman spectroscopy on the myoglobin nitrito heme Fe–O–NO/2-nitrovinyl species in their natural environment rather than in crystals. Our results indicate that the formation of the nitrito heme Fe–O–NO/2-nitrovinyl species is pH-dependent. The conditions under which the nitrito heme Fe–O–NO/2-nitrovinyl species is generated strongly suggest that this form corresponds to an acid induced transformation. We propose that the movement of helices E and F at low pH results in the protonation of nitrito heme Fe–O–NO by His64 Ne–H(E) to form the nitrous heme Fe–O(H)–NO species.

The HO-1/CO system regulates mitochondrial-capillary density relationships in human skeletal muscle.

Abstract: The heme oxygenase-1 (HO-1)/carbon monoxide (CO) system induces mitochondrial biogenesis, but its biological impact in human skeletal muscle is uncertain. The enzyme system generates CO, which stimulates mitochondrial proliferation in normal muscle. Here we examined whether CO breathing can be used to produce a coordinated metabolic and vascular response in human skeletal muscle. In 19 healthy subjects, we performed vastus lateralis muscle biopsies and tested one-legged maximal O2 uptake (Vo2max) before and after breathing air or CO (200 ppm) for 1 h daily for 5 days. In response to CO, there was robust HO-1 induction along with increased mRNA levels for nuclear-encoded mitochondrial transcription factor A (Tfam), cytochrome c, cytochrome oxidase subunit IV (COX IV), and mitochondrial-encoded COX I and NADH dehydrogenase subunit 1 (NDI). CO breathing did not increase Vo2max (1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; P = not significant) but did increase muscle citrate synthase, mitochondrial density (139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO; no. of mitochondrial profiles/field), myoglobin content and glucose transporter (GLUT4) protein level and led to GLUT4 localization to the myocyte membrane, all consistent with expansion of the tissue O2 transport system. These responses were attended by increased cluster of differentiation 31 (CD31)-positive muscle capillaries (1.78 ± 0.16 pre-CO, 2.37 ± 0.59 post-CO; capillaries/muscle fiber), implying the enrichment of microvascular O2 reserve. The findings support that induction of the HO-1/CO system by CO not only improves muscle mitochondrial density, but regulates myoglobin content, GLUT4 localization, and capillarity in accordance with current concepts of skeletal muscle plasticity.

Distal Histidine Modulates the Unusual O-Binding of Nitrite to Myoglobin: Evidence from the Quantum Chemical Analysis of EPR Parameters

Abstract: Nitrite ligand can coordinate with the transition metal through either N- or O-, which is known as linkage isomerism and is believed to occur in metalloproteins. In contrast to the commonly found N-binding motif of nitrite to iron in synthetic models, the less commonly observed O-binding of nitrite to myoglobin (Copeland, D. M.; Soares, A. S.; West, A. H.; Richter-Addo, G. B. J. Inorg. Biol. Chem. 2006, 100, 1413−1425) and hemoglobin (Yi, J.; Safo, M. K.; Richter-Addo, G. Biochemistry, 2008, 47, 8247−8249) reported by Richter-Addo and co-workers is intriguing. On the basis of site-directed mutagenesis studies, it was argued that the distal histidine modulates this unique binding. However, EPR measurements on nitrite binding to methemoglobin could not rule out the possibility of N-bound species to low spin ferric iron. Given to the very similar active sites, there exists a controversy within the two powerful experimental techniques in identifying the coordination motif of nitrite to myoglobin, which is cen...

Structural stability of myoglobin and glycomyoglobin: a comparative molecular dynamics simulation study

Abstract: Glycoproteins are formed as the result of enzymatic glycosylation or chemical glycation in the body, and produced in vitro in industrial processes. The covalently attached carbohydrate molecule(s) confer new properties to the protein, including modified stability. In the present study, the structural stability of a glycoprotein form of myoglobin, bearing a glucose unit in the N-terminus, has been compared with its native form by the use of molecular dynamics simulation. Both structures were subjected to temperatures of 300 and 500 K in an aqueous environment for 10 ns. Changes in secondary structures and RMSD were then assessed. An overall higher stability was detected for glycomyoglobin, for which the most stable segments/residues were highlighted and compared with the native form. The simple addition of a covalently bound glucose is suggested to exert its stabilizing effect via increased contacts with surrounding water molecules, as well as a different pattern of interactions with neighbor residues.