RESUMEN
Heme-containing integral membrane proteins are at the heart of many bioenergetic complexes and electron transport chains. The importance of these electron relay hubs across biology has inspired the design of de novo proteins that recreate their core features within robust, versatile, and tractable protein folds. To this end, we report here the computational design and in-cell production of a minimal diheme membrane cytochrome which successfully integrates into the cellular membrane of live bacteria. This synthetic construct emulates a four-helix bundle found in modern respiratory complexes but has no sequence homology to any polypeptide sequence found in nature. The two b-type hemes, which appear to be recruited from the endogenous heme pool, have distinct split redox potentials with values close to those of natural membrane-spanning cytochromes. The purified protein can engage in rapid biomimetic electron transport with small molecules, with other redox proteins, and with biologically relevant diffusive electron carriers. We thus report an artificial membrane metalloprotein with the potential to serve as a functional electron transfer module in both synthetic protocells and living systems.
Asunto(s)
Citocromos , Metaloproteínas , Citocromos/metabolismo , Oxidación-Reducción , Transporte de Electrón , Metaloproteínas/metabolismo , Hemo/metabolismoRESUMEN
The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.
Asunto(s)
Hemoproteínas , Biofisica , Microscopía por Crioelectrón , Electrones , Oxidación-ReducciónRESUMEN
By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.
Asunto(s)
Biocatálisis , Proteínas de Escherichia coli/química , Metano/análogos & derivados , Peroxidasas/química , Aldehídos/química , Alquenos/química , Aminas/química , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Metano/química , Peroxidasas/metabolismo , Especificidad por SustratoRESUMEN
Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.
Asunto(s)
Proteínas de Escherichia coli , Neoplasias , Profármacos , Humanos , Escherichia coli/metabolismo , Profármacos/química , NAD , Neoplasias/tratamiento farmacológico , Oxidorreductasas , Nitrorreductasas/metabolismo , Proteínas de Escherichia coli/genéticaRESUMEN
NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.
Asunto(s)
Antibacterianos/metabolismo , Benzoquinonas/metabolismo , Proteínas de Escherichia coli/metabolismo , Mononucleótido de Flavina/metabolismo , Nitrofurantoína/metabolismo , Nitrorreductasas/metabolismo , Antibacterianos/química , Benzoquinonas/química , Sitios de Unión/genética , Biocatálisis , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mononucleótido de Flavina/química , Cinética , Simulación de Dinámica Molecular , Estructura Molecular , NADP/metabolismo , Nitrofurantoína/química , Nitrorreductasas/química , Nitrorreductasas/genética , Oxidación-Reducción , Unión Proteica , Dominios Proteicos , Especificidad por SustratoRESUMEN
Natural and artificial proteins with designer properties and functionalities offer unparalleled opportunity for functional nanoarchitectures formed through self-assembly. However, to exploit this potential we need to design the system such that assembly results in desired architecture forms while avoiding denaturation and therefore retaining protein functionality. Here we address this challenge with a model system of fluorescent proteins. By manipulating self-assembly using techniques inspired by soft matter where interactions between the components are controlled to yield the desired structure, we have developed a methodology to assemble networks of proteins of one species which we can decorate with another, whose coverage we can tune. Consequently, the interfaces between domains of each component can also be tuned, with potential applications for example in energy - or electron - transfer. Our model system of eGFP and mCherry with tuneable interactions reveals control over domain sizes in the resulting networks.
Asunto(s)
Nanoestructuras , ProteínasRESUMEN
While proteins have been treated as particles with a spherically symmetric interaction, of course in reality, the situation is rather more complex. A simple step toward higher complexity is to treat the proteins as non-spherical particles and that is the approach we pursue here. We investigate the phase behavior of the enhanced green fluorescent protein (eGFP) under the addition of a non-adsorbing polymer, polyethylene glycol. From small angle x-ray scattering, we infer that the eGFP undergoes dimerization and we treat the dimers as spherocylinders with aspect ratio L/D - 1 = 1.05. Despite the complex nature of the proteins, we find that the phase behavior is similar to that of hard spherocylinders with an ideal polymer depletant, exhibiting aggregation and, in a small region of the phase diagram, crystallization. By comparing our measurements of the onset of aggregation with predictions for hard colloids and ideal polymers [S. V. Savenko and M. Dijkstra, J. Chem. Phys. 124, 234902 (2006) and Lo Verso et al., Phys. Rev. E 73, 061407 (2006)], we find good agreement, which suggests that the behavior of the eGFP is consistent with that of hard spherocylinders and ideal polymers.
Asunto(s)
Coloides , Polímeros , Agregado de Proteínas , Proteínas , Coloides/química , Cristalización , Polímeros/química , Proteínas/químicaRESUMEN
Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light-activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.
Asunto(s)
ADN/química , Luz , Técnicas Analíticas Microfluídicas , Oligonucleótidos/química , Células Artificiales , Fraccionamiento QuímicoRESUMEN
The modification of protein surfaces employing cationic and anionic species enables the assembly of these biomaterials into highly sophisticated hierarchical structures. Such modifications can allow bioconjugates to retain or amplify their functionalities under conditions in which their native structure would be severely compromised. In this work, we assess the effect of this type of bioconjugation on the redox properties of two model heme proteins, that is, cytochrome c (CytC) and myoglobin (Mb). In particular, the work focuses on the sequential modification by 3-dimethylamino propylamine (DMAPA) and 4-nonylphenyl 3-sulfopropyl ether (S1) anionic surfactant. Bioconjugation with DMAPA and S1 are the initial steps in the generation of pure liquid proteins, which remain active in the absence of water and up to temperatures above 150 °C. Thin-layer spectroelectrochemistry reveals that DMAPA cationization leads to a distribution of bioconjugate structures featuring reduction potentials shifted up to 380 mV more negative than the native proteins. Analysis based on circular dichroism, MALDI-TOF mass spectrometry, and zeta potential measurements suggest that the shift in the reduction potentials are not linked to protein denaturation, but to changes in the spin state of the heme. These alterations of the spin states originate from subtle structural changes induced by DMAPA attachment. Interestingly, electrostatic coupling of anionic surfactant S1 shifts the reduction potential closer to that of the native protein, demonstrating that the modifications of the heme electronic configuration are linked to surface charges.
Asunto(s)
Citocromos c/química , Hemo/química , Mioglobina/química , Aniones/química , Arsenicales/química , Dicroismo Circular , Oxidación-Reducción , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica , Electricidad Estática , Temperatura , Agua/químicaRESUMEN
Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.
Asunto(s)
Oxidorreductasas/metabolismo , Proteínas/química , Hemo/química , Hemo/metabolismo , Modelos Moleculares , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidorreductasas/química , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas/métodosRESUMEN
The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.
Asunto(s)
Proteínas Portadoras/síntesis química , Proteínas Portadoras/metabolismo , Oxígeno/metabolismo , Ingeniería de Proteínas , Transporte Biológico , Monóxido de Carbono/metabolismo , Proteínas Portadoras/química , Diseño de Fármacos , Globinas/química , Ácido Glutámico/metabolismo , Hemo/metabolismo , Histidina/metabolismo , Humanos , Cinética , Ligandos , Proteínas del Tejido Nervioso/química , Neuroglobina , Oxidación-Reducción , Estructura Secundaria de Proteína , Rotación , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por Sustrato , Agua/análisis , Agua/metabolismoRESUMEN
The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.
Asunto(s)
Calcio/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Complejo Cetoglutarato Deshidrogenasa/genética , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Miocardio/enzimología , Unión Proteica , Subunidades de Proteína , Especificidad de la Especie , PorcinosRESUMEN
Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.
Asunto(s)
Hemo , Oxidación-Reducción , Hemo/química , Hemo/metabolismo , Solubilidad , Agua/química , Agua/metabolismo , Citocromos/química , Citocromos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Electricidad Estática , Ingeniería de ProteínasRESUMEN
Synthetic cannabinoid receptor agonists (SCRAs) are one of the fastest growing classes of recreational drugs. Despite their growth in use, their vast chemical diversity and rapidly changing landscape of structures make understanding their effects challenging. In particular, the side effects for SCRA use are extremely diverse, but notably include severe outcomes such as cardiac arrest. These side effects appear at odds with the main putative mode of action, as full agonists of cannabinoid receptors. We have hypothesized that SCRAs may act as MAO inhibitors, owing to their structural similarity to known monoamine oxidase inhibitors (MAOI's) as well as matching clinical outcomes (hypertensive crisis) of 'monoaminergic toxicity' for users of MAOIs and some SCRA use. We have studied the potential for SCRA-mediated inhibition of MAO-A and MAO-B via a range of SCRAs used commonly in the UK, as well as structural analogues to prove the atomistic determinants of inhibition. By combining in silico and experimental kinetic studies we demonstrate that SCRAs are MAO-A-specific inhibitors and their affinity can vary significantly between SCRAs, most notably affected by the nature of the SCRA 'head' group. Our data allow us to posit a putative mechanism of inhibition. Crucially our data demonstrate that SCRA activity is not limited to just cannabinoid receptor agonism and that alternative interactions might account for some of the diversity of the observed side effects and that these effects can be SCRA-specific.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Drogas Ilícitas , Agonistas de Receptores de Cannabinoides/farmacología , Agonistas de Receptores de Cannabinoides/química , Cinética , Inhibidores de la Monoaminooxidasa/farmacología , MonoaminooxidasaRESUMEN
The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.
Asunto(s)
Oxidorreductasas/síntesis química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/síntesis química , Biología Sintética/métodos , Oxidación-Reducción , Oxidorreductasas/química , Proteínas Recombinantes/químicaRESUMEN
Tetrapyrrole cofactors such as heme and chlorophyll imprint their intrinsic reactivity and properties on a multitude of natural proteins and enzymes, and there is much interest in exploiting their functional and catalytic capabilities within minimal, de novo designed protein scaffolds. Here we describe how, using only natural biosynthetic and post-translational modification pathways, de novo designed soluble and hydrophobic proteins can be equipped with tetrapyrrole cofactors within living Escherichia coli cells. We provide strategies to achieve covalent and non-covalent heme incorporation within the de novo proteins and describe how the heme biosynthetic pathway can be co-opted to produce the light sensitive zinc protoporphyrin IX for loading into proteins in vivo. In addition, we describe the imaging of hydrophobic proteins and cofactor-rich protein droplets by electron and fluorescence microscopy, and how cofactors can be stripped from the de novo proteins to aid in vitro identification.
Asunto(s)
Proteínas/metabolismo , Clorofila , Escherichia coli/genética , Hemo , Proteínas/genética , TetrapirrolesRESUMEN
We report the mutational analysis of an artificial oxygen transport protein, HP7, which operates via a mechanism akin to that of human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of 13. Paradoxically, it also decreases heme binding affinity by a factor of 5 in the reduced state and 60 in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates for that. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins.
Asunto(s)
Genes Sintéticos , Hemo/metabolismo , Hemoproteínas/química , Secuencia de Aminoácidos , Secuencia de Bases , Hemo/química , Hemoproteínas/síntesis química , Hemoproteínas/genética , Hemoproteínas/metabolismo , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Oxígeno/metabolismo , Unión Proteica , Ingeniería de Proteínas , TermodinámicaRESUMEN
Here we extend the engineering descriptions of simple, single-electron-tunneling chains common in oxidoreductases to quantify sequential oxidation-reduction rates of two-or-more electron cofactors and substrates. We identify when nicotinamides may be vulnerable to radical mediated oxidation-reduction and merge electron-tunneling expressions with the chemical rate expressions of Eyring. The work provides guidelines for the construction of new artificial oxidoreductases inspired by Nature but adopting independent design and redox engineering.
Asunto(s)
Transporte de Electrón , Oxidorreductasas/química , Oxidación-Reducción , Fotosíntesis , Ingeniería de ProteínasRESUMEN
De novo enzymes can be created by computational design and directed evolution. Here, we review recent insights into the origins of catalytic power in evolved designer enzymes to pinpoint opportunities for next-generation designs: Evolution precisely organizes active sites, introduces catalytic H-bonding networks, invokes electrostatic catalysis, and creates dynamical networks embedding the active site in a reactive protein scaffold. Such insights foster our fundamental knowledge of enzyme catalysis and fuel the future design of tailor-made enzymes.
Asunto(s)
Evolución Molecular Dirigida , Enzimas , Proteínas , Catálisis , Dominio Catalítico , Enzimas/genética , Enzimas/metabolismo , Ingeniería de Proteínas , Proteínas/genética , Electricidad EstáticaRESUMEN
Activation heat capacity is emerging as a crucial factor in enzyme thermoadaptation, as shown by the non-Arrhenius behaviour of many natural enzymes. However, its physical origin and relationship to the evolution of catalytic activity remain uncertain. Here we show that directed evolution of a computationally designed Kemp eliminase reshapes protein dynamics, which gives rise to an activation heat capacity absent in the original design. These changes buttress transition-state stabilization. Extensive molecular dynamics simulations show that evolution results in the closure of solvent-exposed loops and a better packing of the active site. Remarkably, this gives rise to a correlated dynamical network that involves the transition state and large parts of the protein. This network tightens the transition-state ensemble, which induces a negative activation heat capacity and non-linearity in the activity-temperature dependence. Our results have implications for understanding enzyme evolution and suggest that selectively targeting the conformational dynamics of the transition-state ensemble by design and evolution will expedite the creation of novel enzymes.