-
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 -
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 -
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 -
Molecular Aspects of Medicine Apr 2022Antonini and Brunori's 1971 book "Hemoglobin and Myoglobin in Their Reactions with Ligands" was a truly remarkable publication that summarized almost 100 years of... (Review)
Review
Antonini and Brunori's 1971 book "Hemoglobin and Myoglobin in Their Reactions with Ligands" was a truly remarkable publication that summarized almost 100 years of research on O binding to these globins. Over the ensuing 50 years, ultra-fast laser photolysis techniques, high-resolution and time resolved X-ray crystallography, molecular dynamics simulations, and libraries of recombinant myoglobin (Mb) and hemoglobin (Hb) variants have provided structural interpretations of O binding to these proteins. The resultant mechanisms provide quantitative descriptions of the stereochemical factors that govern overall affinity, including proximal and distal steric restrictions that affect iron reactivity and favorable positive electrostatic interactions that preferentially stabilize bound O. The pathway for O uptake and release by Mb and subunits of Hb has been mapped by screening libraries of site-directed mutants in laser photolysis experiments. O enters mammalian Mb and the α and β subunits of human HbA through a channel created by upward and outward rotation of the distal His at the E7 helical position, is non-covalently captured in the interior of the distal cavity, and then internally forms a bond with the heme Fe(II) atom. O dissociation is governed by disruption of hydrogen bonding interactions with His (E7), breakage of the Fe(II)-O bond, and then competition between rebinding and escape through the E7-gate. The structural features that govern the rates of both the individual steps and overall reactions have been determined and provide the framework for: (1) defining the physiological functions of specific globins and their evolution; (2) understanding the clinical features of hemoglobinopathies; and (3) designing safer and more efficient acellular hemoglobin-based oxygen carriers (HBOCs) for transfusion therapy, organ preservation, and other commercially relevant O transport and storage processes.
Topics: Animals; Carbon Monoxide; Hemoglobins; Humans; Kinetics; Ligands; Mammals; Myoglobin; Oxygen
PubMed: 34544605
DOI: 10.1016/j.mam.2021.101024 -
Protein Science : a Publication of the... Feb 2010Over the last half century, myoglobin (Mb) has been an excellent model system to test a number of concepts, theories, and new experimental methods that proved valuable... (Review)
Review
Over the last half century, myoglobin (Mb) has been an excellent model system to test a number of concepts, theories, and new experimental methods that proved valuable to investigate protein structure, function, evolution, and dynamics. Mb's function, most often considered just an oxygen repository, has considerably diversified over the last 15 years, especially because it was shown to have a role in the biochemistry of quenching and synthesizing nitric oxide in the red muscle, thereby protecting the cell. To tackle protein's structural dynamics by innovative biophysical methods, Mb has been the best prototype; laser flash technology made it possible to obtain molecular movies by time-resolved Laue crystallography (with ps resolution). This approach unveiled the complexity of the energy landscape and the structural basis of the stretched interconversion between conformational substates of a protein.
Topics: Animals; Heme; Humans; Myoglobin; Oxygen; Protein Conformation; Substrate Specificity
PubMed: 19953516
DOI: 10.1002/pro.300 -
The Journal of Experimental Biology Aug 2010Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers... (Review)
Review
Myoglobin, a mobile carrier of oxygen, is without a doubt an important player central to the physiological function of heart and skeletal muscle. Recently, researchers have surmounted technical challenges to measure Mb diffusion in the living cell. Their observations have stimulated a discussion about the relative contribution made by Mb-facilitated diffusion to the total oxygen flux. The calculation of the relative contribution, however, depends upon assumptions, the cell model and cell architecture, cell bioenergetics, oxygen supply and demand. The analysis suggests that important differences can be observed whether steady-state or transient conditions are considered. This article reviews the current evidence underlying the evaluation of the biophysical parameters of myoglobin-facilitated oxygen diffusion in cells, specifically the intracellular concentration of myoglobin, the intracellular diffusion coefficient of myoglobin and the intracellular myoglobin oxygen saturation. The review considers the role of myoglobin in oxygen transport in vertebrate heart and skeletal muscle, in the diving seal during apnea as well as the role of the analogous leghemoglobin of plants. The possible role of myoglobin in intracellular fatty acid transport is addressed. Finally, the recent measurements of myoglobin diffusion inside muscle cells are discussed in terms of their implications for cytoarchitecture and microviscosity in these cells and the identification of intracellular impediments to the diffusion of proteins inside cells. The recent experimental data then help to refine our understanding of Mb function and establish a basis for future investigation.
Topics: Animals; Diffusion; Diving; Fatty Acids; Humans; Muscle, Skeletal; Myocardium; Myoglobin; Nuclear Magnetic Resonance, Biomolecular; Oxygen; Oxygen Consumption
PubMed: 20675540
DOI: 10.1242/jeb.043075 -
Antioxidants & Redox Signaling Jun 2013Failure to maintain myoglobin (Mb) in the reduced state causes the formation of metMb, ferryl Mb species, and cross-linked Mb. Dissociation of ferriprotoporphyrin IX... (Review)
Review
SIGNIFICANCE
Failure to maintain myoglobin (Mb) in the reduced state causes the formation of metMb, ferryl Mb species, and cross-linked Mb. Dissociation of ferriprotoporphyrin IX from the globin and release of iron atoms can also occur as oxidized Mb accumulates. These modifications may contribute to various oxidative pathologies in muscle and muscle foods.
RECENT ADVANCES
The mechanism of ferryl Mb-mediated oxidative damage to nearby structures has been partially elucidated. Dissociation of ferriprotoporphyrin IX from metMb occurs more readily at acidic pH values. The dissociated ferriprotoporphyrin IX (also called hemin) readily decomposes preformed lipid hydroperoxides to reactive oxygen species. Heme oxygenase as well as lipophilic free radicals can degrade the protoporphyrin IX moiety, which results in the formation of free iron.
CRITICAL ISSUES
The multiple pathways by which Mb can incur toxicity create difficulties in determining the major cause of oxidative damage in a particular system. Peroxides and low pH activate each of the oxidative Mb forms, ferriprotoporphyrin IX, and released iron. Determining the relative concentration of these species is technically difficult, but essential to a complete understanding of oxidative pathology in muscle tissue.
FUTURE DIRECTIONS
Improved methods to assess the different pathways of Mb toxicity are needed. Although significant advances have been made in the understanding of Mb interactions with other biomolecules, further investigation is needed to understand the physical and chemical nature of these interactions.
Topics: Hemin; Humans; Iron; Myoglobin; Oxidation-Reduction; Peroxides; Protein Binding
PubMed: 22900975
DOI: 10.1089/ars.2012.4887 -
The Journal of Biological Chemistry 2021Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions.... (Review)
Review
Proteins are the molecular machines of living systems. Their dynamics are an intrinsic part of their evolutionary selection in carrying out their biological functions. Although the dynamics are more difficult to observe than a static, average structure, we are beginning to observe these dynamics and form sound mechanistic connections between structure, dynamics, and function. This progress is highlighted in case studies from myoglobin and adenylate kinase to the ribosome and molecular motors where these molecules are being probed with a multitude of techniques across many timescales. New approaches to time-resolved crystallography are allowing simple "movies" to be taken of proteins in action, and new methods of mapping the variations in cryo-electron microscopy are emerging to reveal a more complete description of life's machines. The results of these new methods are aided in their dissemination by continual improvements in curation and distribution by the Protein Data Bank and their partners around the world.
Topics: Adenylate Kinase; Animals; Databases, Protein; Humans; Models, Molecular; Myoglobin; Ribosomes; Structure-Activity Relationship
PubMed: 33961840
DOI: 10.1016/j.jbc.2021.100749 -
Antioxidants & Redox Signaling Feb 2020Over the past 50 years, the mechanisms for O storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and... (Review)
Review
Over the past 50 years, the mechanisms for O storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and timescales from seconds to picoseconds. In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O uptake by mammalian red cells is limited by diffusion through unstirred water layers adjacent to the cell surface and across cell-free layers adjacent to vessel walls. (ii) In most vertebrates, hemoglobins (Hbs) and myoglobins (Mbs), the distal histidine at the E7 helical position donates a strong hydrogen bond to bound O, which selectively enhances O affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O binding to mammalian Hbs and Mbs occurs by migration of the ligand through a channel created by upward rotation of the His(E7) side chain, capture in the empty space of the distal pocket, and then coordination with the ferroprotoporphyrin IX (heme) iron atom. (iv) The assembly of Mbs and Hbs occurs by formation of molten globule intermediates, in which the N- and C-terminal helices have almost fully formed secondary structures, but the heme pockets are disordered and followed by high-affinity binding of heme. These conclusions indicate that there are often compromises between O transport function, holoprotein stability, and the efficiency of assembly. However, the biochemical mechanisms underlying these conclusions provide the framework for understanding globin evolution in greater detail and for engineering more efficient and stable globins.
Topics: Animals; Hemoglobins; Humans; Models, Molecular; Myoglobin; Oxygen; Protein Stability; Protein Structure, Secondary; Static Electricity
PubMed: 31530172
DOI: 10.1089/ars.2019.7876 -
Journal of the American Chemical Society Mar 2015We use Markovian milestoning molecular dynamics (MD) simulations on a tessellation of the collective variable space for CO localization in myoglobin to estimate the...
We use Markovian milestoning molecular dynamics (MD) simulations on a tessellation of the collective variable space for CO localization in myoglobin to estimate the kinetics of entry, exit, and internal site-hopping. The tessellation is determined by analysis of the free-energy surface in that space using transition-path theory (TPT), which provides criteria for defining optimal milestones, allowing short, independent, cell-constrained MD simulations to provide properly weighted kinetic data. We coarse grain the resulting kinetic model at two levels: first, using crystallographically relevant internal cavities and their predicted interconnections and solvent portals; and second, as a three-state side-path scheme inspired by similar models developed from geminate recombination experiments. We show semiquantitative agreement with experiment on entry and exit rates and in the identification of the so-called "histidine gate" at position 64 through which ≈90% of flux between solvent and the distal pocket passes. We also show with six-dimensional calculations that the minimum free-energy pathway of escape through the histidine gate is a "knock-on" mechanism in which motion of the ligand and the gate are sequential and interdependent. In total, these results suggest that such TPT simulations are indeed a promising approach to overcome the practical time-scale limitations of MD to allow reliable estimation of transition mechanisms and rates among metastable states.
Topics: Binding Sites; Carbon Monoxide; Diffusion; Kinetics; Ligands; Models, Molecular; Myoglobin; Protein Conformation
PubMed: 25664858
DOI: 10.1021/ja512484q