Natural gas stoves in >40 million U.S. residences release methane (CH4)─a potent greenhouse gas─through post-meter leaks and incomplete combustion. We quantified methane released in 53 homes during ...all phases of stove use: steady-state-off (appliance not in use), steady-state-on (during combustion), and transitory periods of ignition and extinguishment. We estimated that natural gas stoves emit 0.8–1.3% of the gas they use as unburned methane and that total U.S. stove emissions are 28.1 95% confidence interval: 18.5, 41.2 Gg CH4 year–1. More than three-quarters of methane emissions we measured originated during steady-state-off. Using a 20-year timeframe for methane, annual methane emissions from all gas stoves in U.S. homes have a climate impact comparable to the annual carbon dioxide emissions of 500 000 cars. In addition to methane emissions, co-emitted health-damaging air pollutants such as nitrogen oxides (NOx) are released into home air and can trigger respiratory diseases. In 32 homes, we measured NOx (NO and NO2) emissions and found them to be linearly related to the amount of natural gas burned (r2 = 0.76; p ≪ 0.01). Emissions averaged 21.7 20.5, 22.9 ng NOx J–1, comprised of 7.8 7.1, 8.4 ng NO2 J–1 and 14.0 12.8, 15.1 ng NO J–1. Our data suggest that families who don't use their range hoods or who have poor ventilation can surpass the 1-h national standard of NO2 (100 ppb) within a few minutes of stove usage, particularly in smaller kitchens.
Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. ...The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein ...reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.
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•Glutamine-binding proteins evolved from promiscuous arginine-binding proteins•Conformational plasticity and water networks enabled promiscuous Gln binding•An alternative Gln-binding conformation was observed in the Arg-bound structure•A reduction of the entropic penalty to binding resulted in increased affinity for Gln
Proteins can evolve new functions through improvement of latent promiscuous functions. In this work, Clifton and Jackson present a structural and thermodynamic analysis of ancestral solute-binding proteins, highlighting the roles of conformational plasticity and enthalpic interactions in a promiscuous protein-ligand complex that enabled the evolution of high-affinity glutamine-binding proteins.
5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, ...5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.
Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory ...circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.
•Induction of synaptic LTP prompts withdrawal of perisynaptic astroglia•The underlying mechanisms involve NKCC1 transporter and cofilin•Reduced synaptic astroglial coverage boosts extrasynaptic glutamate escape•LTP induction thus enhances NMDAR-dependent inter-synaptic cross-talk
Central synapses are often surrounded by thin astroglial processes that confine chemical neurotransmission to the synaptic cleft. Henneberger et al. find that memory trace formation at synaptic connections prompts withdrawal of these processes, thus boosting extrasynaptic neurotransmitter actions. Such actions can alter signal integration rules among neighboring synapses.
In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins ...generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families. As ASR becomes more widely adopted as a protein engineering approach, it is important to understand the applications, limitations, and recent developments of this technique. This review highlights recent exemplifications of ASR, as well as technical aspects of the reconstruction process that are relevant to protein engineering.
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•Proteins produced by ancestral sequence reconstruction (ASR) often exhibit high activity, stability, and evolvability.•ASR studies guide engineering by highlighting function-shifting substitutions.•ASR emphasizes fundamental drivers of, and constraints to, functional innovation.•Recent developments in ASR are making it more accessible to protein engineers.
•Laboratory directed evolution and ancestral reconstruction can facilitate study of protein structural dynamics.•Advances in biophysical and computational techniques facilitate this ...investigation.•Conformational dynamism is valuable in the acquisition of new function, as multiple potentially productive conformations can be sampled.•Evolution ‘freezes out’ unproductive motions, reducing unnecessary dynamics.
This review focuses on recent work that has begun to establish specific functional roles for protein conformational dynamics, specifically how the conformational landscapes that proteins can sample can evolve under laboratory based evolutionary selection. We discuss recent technical advances in computational and biophysical chemistry, which have provided us with new ways to dissect evolutionary processes. Finally, we offer some perspectives on the emerging view of conformational dynamics and evolution, and the challenges that we face in rationally engineering conformational dynamics.
Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3′-phosphoadenosine ...5′-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity comparedwith its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.
Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known ...relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677–13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE’s active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k cat/K M values of up to 2 × 104). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE’s paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.
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