Imaging of gene-expression patterns in live animals is difficult to achieve with fluorescent proteins because tissues are opaque to visible light. Imaging of transgene expression with magnetic ...resonance imaging (MRI), which penetrates to deep tissues, has been limited by single reporter visualization capabilities. Moreover, the low-throughput capacity of MRI limits large-scale mutagenesis strategies to improve existing reporters. Here we develop an MRI system, called GeneREFORM, comprising orthogonal reporters for two-color imaging of transgene expression in deep tissues. Starting from two promiscuous deoxyribonucleoside kinases, we computationally designed highly active, orthogonal enzymes ('reporter genes') that specifically phosphorylate two MRI-detectable synthetic deoxyribonucleosides ('reporter probes'). Systemically administered reporter probes exclusively accumulate in cells expressing the designed reporter genes, and their distribution is displayed as pseudo-colored MRI maps based on dynamic proton exchange for noninvasive visualization of transgene expression. We envision that future extensions of GeneREFORM will pave the way to multiplexed deep-tissue mapping of gene expression in live animals.
Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve ...tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.
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•Computational design stabilized human and bovine serum albumins.•Designs express solubly in E. coli and exhibit up to 40 °C increased thermostability.•Some designs exhibit identical ...ligand binding properties.•Crystal structure confirms design accuracy.•Designs can be used in cell culture and in vitro applications.
Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.
A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced ...by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.
NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and ...stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 Å resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here ...we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
Protein complexes often represent an ensemble of different assemblies with distinct functions and regulation. This increased complexity is enabled by the variety of protein diversification mechanisms ...that exist at every step of the protein biosynthesis pathway, such as alternative splicing and post transcriptional and translational modifications. The resulting variation in subunits can generate compositionally distinct protein assemblies. These different forms of a single protein complex may comprise functional variances that enable response and adaptation to varying cellular conditions. Despite the biological importance of this layer of complexity, relatively little is known about the compositional heterogeneity of protein complexes, mostly due to technical barriers of studying such closely related species. Here, we show that native mass spectrometry (MS) offers a way to unravel this inherent heterogeneity of protein assemblies. Our approach relies on the advanced Orbitrap mass spectrometer capable of multistage MS analysis across all levels of protein organization. Specifically, we have implemented a two-step fragmentation process in the inject flatapole device, which was converted to a linear ion trap, and can now probe the intact protein complex assembly, through its constituent subunits, to the primary sequence of each protein. We demonstrate our approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.
Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological ...functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.
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•Our work suggest that during translation of the SRP receptor FtsY, its N-domain (helices N2–4) assumes a distinct functional folding intermediate, which then disappears after completion of the receptor translation. This is based on new crystal structures and various biochemical results.•Our work presents evidence that upon completion of FtsY translation and its release from the ribosome, residues at the C-terminus of the G-domain facilitate the formation of a stable, three-helical bundle state of N2–4.•The conformational switch of N2–4 might separate between its role in cotranslational targeting of FtsY and ribosomes to the cytoplasmic membrane and its role in the assembly and downstream functions of the mature receptor.
Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. ...However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme—β-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP–free grass pea cultivars, which are safe for human and animal consumption.
Abstract
Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and ...the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.
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