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Journal of the American Chemical Society Feb 2022The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical...
The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.
Topics: Aeromonas hydrophila; Bacterial Toxins; Cytochromes c; Hemolysin Proteins; Muramidase; Myoglobin; Nanopores; Peptide Fragments; Pore Forming Cytotoxic Proteins; Proteolysis; Proteomics; Trypsin
PubMed: 35120294
DOI: 10.1021/jacs.1c11758 -
Nature Oct 2022Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins. Nonetheless, its potential in even...
Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins. Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins. Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations. Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme. Machine learning methods have recently showed promise in predicting productive mutations, but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution.
Topics: Biocatalysis; Catalytic Domain; Directed Molecular Evolution; Magnetic Resonance Spectroscopy; Mutation; Myoglobin; Oxygen
PubMed: 36198791
DOI: 10.1038/s41586-022-05278-9 -
Journal of Proteomics Jun 2023Color of retail fresh beef is the most important quality influencing the consumers' purchase decisions at the point of sale. Discolored fresh beef cuts are either... (Review)
Review
Color of retail fresh beef is the most important quality influencing the consumers' purchase decisions at the point of sale. Discolored fresh beef cuts are either discarded or converted to low-value products, before the microbial quality is compromised, resulting in huge economic loss to meat industry. The interinfluential interactions between myoglobin, small biomolecules, proteome, and cellular components in postmortem skeletal muscles govern the color stability of fresh beef. This review examines the novel applications of high-throughput tools in mass spectrometry and proteomics to elucidate the fundamental basis of these interactions and to explain the underpinning mechanisms of fresh beef color. Advanced proteomic research indicates that a multitude of factors endogenous to skeletal muscles critically influence the biochemistry of myoglobin and color stability in fresh beef. Additionally, this review highlights the potential of muscle proteome components and myoglobin modifications as novel biomarkers for fresh beef color. SIGNIFICANCE: This review highlights the important role of muscle proteome in fresh beef color, which is the major trait impacting consumers' purchase decisions. In recent years, innovative approaches in proteomics have been exploited for an in-depth understanding of the biochemical mechanisms influencing color development and color stability in fresh beef. The review suggests that a wide range of factors, including endogenous skeletal muscle components, can affect myoglobin biochemistry and color stability in beef. Furthermore, the potential use of muscle proteome components and myoglobin post-translational modifications as biomarkers for fresh beef color is discussed. The currently available body of evidence presented in this review can have important implications in meat industry as it provides novel insights into the factors influencing fresh beef color and an up-to-date list of biomarkers that can be used to predict beef color quality.
Topics: Animals; Cattle; Myoglobin; Proteomics; Proteome; Meat; Muscle, Skeletal; Color
PubMed: 37024077
DOI: 10.1016/j.jprot.2023.104893 -
Molecular Aspects of Medicine Apr 2022Under those pathological conditions in which Myoglobin and Hemoglobin escape their cellular environments and are thus separated from cellular reductive/protective... (Review)
Review
Under those pathological conditions in which Myoglobin and Hemoglobin escape their cellular environments and are thus separated from cellular reductive/protective systems, the inherent peroxidase activities of these proteins can be expressed. This activity leads to the formation of the highly oxidizing oxo-ferryl species. Evidence that this happens in vivo is provided by the formation of a covalent bond between the heme group and the protein and this acts as an unambiguous biomarker for the presence of the oxo ferryl form. The peroxidatic activity also leads to the oxidation of lipids, the products of which can be powerful vasoconstrictive agents (e.g. isoprostanes, neuroprostanes). Here we review the evidence that lipid oxidation occurs following rhabdomyolysis and sub-arachnoid hemorrhage and that the products formed from arachidonic acid chains of phospholipids lead, through vasoconstriction, to kidney failure and brain vasospasm. Intervention in these pathological conditions through administration of reducing agents to remove ferryl heme is discussed. Through-protein electron transfer pathways that facilitate ferryl reduction at low reductant concentration have been identified. We conclude with consideration of the therapeutic use of Hemoglobin Based Oxygen carriers and how the toxicity of these may be reduced by engineering such electron transfer pathways into hemoglobin.
Topics: Heme; Hemoglobins; Humans; Myoglobin; Oxidation-Reduction; Oxygen
PubMed: 34654576
DOI: 10.1016/j.mam.2021.101045 -
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 -
Angewandte Chemie (International Ed. in... Dec 2018Carbon bonds (C-bonds) are the highly directional noncovalent interactions between carbonyl-oxygen acceptors and sp -hybridized-carbon σ-hole donors through n→σ*...
Carbon bonds (C-bonds) are the highly directional noncovalent interactions between carbonyl-oxygen acceptors and sp -hybridized-carbon σ-hole donors through n→σ* electron delocalization. We have shown the ubiquitous existence of C-bonds in proteins with the help of careful protein structure analysis and quantum calculations, and have precisely determined C-bond energies. The importance of conventional noncovalent interactions such as hydrogen bond (H-bonds) and halogen bond (X-bonds) in the structure and function of biological molecules are well established, while carbon bonds C-bonds have still to be recognized. We have shown that C-bonds are present in proteins, contribute enthalpically to the overall hydrophobic interaction and play a significant role in the photodissociation mechanism of myoglobin and the binding of nucleobases to proteins.
Topics: Animals; Carbon; Horses; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Myoglobin; Proteins; Quantum Theory; Thermodynamics
PubMed: 30347500
DOI: 10.1002/anie.201811171 -
Journal of Molecular Biology Aug 2017Sixty years ago, the first protein structure of myoglobin was determined by John Kendrew and his colleagues; hemoglobin followed shortly thereafter. For quite some time,... (Review)
Review
Sixty years ago, the first protein structure of myoglobin was determined by John Kendrew and his colleagues; hemoglobin followed shortly thereafter. For quite some time, it seemed that only X-ray crystallography would be capable of determining the structure of proteins to high resolution. In recent years, cryo-electron microscopy has emerged as a viable alternative and indeed in many cases the preferred approach. It is capable of studying proteins that span a size range from several megadaltons to proteins as small as myoglobin and hemoglobin.
Topics: Cryoelectron Microscopy; Hemoglobins; Myoglobin; Protein Conformation
PubMed: 28697886
DOI: 10.1016/j.jmb.2017.07.004 -
American Journal of Physiology.... Jul 2021Myoglobin (Mb) is a regulator of O bioavailability in type I muscle and heart, at least when tissue O levels drop. Mb also plays a role in regulating cellular nitric...
Myoglobin (Mb) is a regulator of O bioavailability in type I muscle and heart, at least when tissue O levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection. To evaluate this hypothesis, we measured energy expenditure (EE), respiratory exchange ratio (RER), body weight gain and adiposity, glucose tolerance, and insulin sensitivity in Mb knockout (Mb) and wild-type (WT) mice challenged with a high-fat diet (HFD, 45% of calories). In males ( = 10/genotype) and females ( = 9/genotype) tested at 5-6, 11-12, and 17-18 wk, there were no genotype effects on RER, EE, or food intake. RER and EE during cold (10°C, 72 h), and glucose and insulin tolerance, were not different compared with within-sex WT controls. At ∼18 and ∼19 wk of age, female Mb adiposity was ∼42%-48% higher versus WT females ( = 0.1). Transcriptomics analyses (whole gastrocnemius, soleus) revealed few consistent changes, with the notable exception of a 20% drop in soleus transferrin receptor (Tfrc) mRNA. Capillarity indices were significantly increased in Mb, specifically in Mb-rich soleus and deep gastrocnemius. The results indicate that Mb loss does not have a major impact on whole body glucose homeostasis, EE, RER, or response to a cold challenge in mice. However, the greater adiposity in female Mb mice indicates a sex-specific effect of Mb KO on fat storage and feed efficiency. The roles of myoglobin remain to be elaborated. We address sexual dimorphism in terms of outcomes in response to the loss of myoglobin in knockout mice and perform, for the first time, a series of comprehensive metabolic studies under conditions in which fat is mobilized (high-fat diet, cold). The results highlight that myoglobin is not necessary and sufficient for maintaining oxidative metabolism and point to alternative roles for this protein in muscle and heart.
Topics: Adiposity; Animals; Body Weight; Diet, High-Fat; Energy Metabolism; Fatty Acids; Female; Glucose Tolerance Test; Lipid Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Myocardium; Myoglobin; Oxidation-Reduction; Phenotype; Sex Characteristics
PubMed: 33969704
DOI: 10.1152/ajpendo.00624.2020 -
Journal of Agricultural and Food... Nov 2020Appearance is an important sensory property that significantly influences consumers' perceptions of fresh meat quality. Failure to meet consumer expectations can lead to... (Review)
Review
Appearance is an important sensory property that significantly influences consumers' perceptions of fresh meat quality. Failure to meet consumer expectations can lead to rejection of meat products, concomitant loss in value, and potential production of organic waste. Immediately after animal harvest, skeletal muscle metabolism changes from aerobic to anaerobic. However, anoxic post-mortem muscle is biochemically active, and biomolecular interaction between myoglobin, mitochondria, metabolites, and lipid oxidation determines meat color. This review examines how metabolites and mitochondrial activity can influence myoglobin oxygenation and metmyoglobin reducing activity. Further, the review highlights recent research that has examined myoglobin redox dynamics, sarcoplasmic metabolite changes, and/or post-mortem biochemistry.
Topics: Animals; Color; Lipids; Meat; Muscle, Skeletal; Myoglobin; Oxidation-Reduction; Postmortem Changes
PubMed: 32045229
DOI: 10.1021/acs.jafc.9b08098 -
Comprehensive Reviews in Food Science... Jul 2023Meat color is an important aspect for the meat industry since it strongly determines the consumers' perception of product quality and thereby significantly influences... (Review)
Review
Meat color is an important aspect for the meat industry since it strongly determines the consumers' perception of product quality and thereby significantly influences the purchase decision. Emergence of new vegan meat analogs has renewed interest in the fundamental aspects of meat color in order to replicate it. The appearance of meat is based on a complex interplay between the pigment-based meat color from myoglobin and its chemical forms and light scattering from the muscle's microstructure. While myoglobin biochemistry and pigment-based meat color have been extensively studied, research on the physicochemical contribution of light scattering to meat color and the special case of structural colors causing meat iridescence has received only little attention. Former review articles focused mostly on the biochemical or physical mechanisms rather than the interplay between them, in particular the role that structural colors play. While from an economic point of view, meat iridescence might be considered negligible, an enhanced understanding of the underlying mechanisms and the interactions of light with meat microstructures can improve our overall understanding of meat color. Therefore, this review discusses both biochemical and physicochemical aspects of meat color including the origin of structural colors, highlights new color measurement methodologies suitable to investigate color phenomena such as meat iridescence, and finally presents approaches to modulate meat color in terms of base composition, additives, and processing.
Topics: Myoglobin; Iridescence; Meat; Color; Consumer Behavior
PubMed: 37306532
DOI: 10.1111/1541-4337.13191