ABSTRACT
Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.
Subject(s)
Indenes , Mixed Function Oxygenases , Oxidation-Reduction , Hydroxylation , BiocatalysisABSTRACT
InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to â¼3200-fold. Replacement of Y158 with 3-fluoroTyr (3-FY) and 3,5-difluoroTyr (3,5-F2Y) has no effect on kcatapp/KMapp nor on the binding of inhibitors to the open form of the enzyme (Kiapp), whereas both kcatapp/KMapp and Kiapp are altered by seven-fold for the 2,3,5-trifluoroTyr variant (2,3,5-F3Y158 InhA). 19F NMR spectroscopy suggests that 2,3,5-F3Y158 is ionized at neutral pH indicating that neither the acidity nor ionization state of residue 158 has a major impact on catalysis or on the binding of substrate-like inhibitors. In contrast, Ki*app is decreased 6- and 35-fold for the binding of the slow-onset inhibitor PT504 to 3,5-F2Y158 and 2,3,5-F3Y158 InhA, respectively, indicating that Y158 stabilizes the closed form of the enzyme adopted by EI*. The residence time of PT504 is reduced â¼four-fold for 2,3,5-F3Y158 InhA compared to wild-type, and thus, the hydrogen bonding interaction of the inhibitor with Y158 is an important factor in the design of InhA inhibitors with increased residence times on the enzyme.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Isoniazid/chemistry , Isoniazid/pharmacology , Catalytic Domain , Bacterial Proteins/chemistryABSTRACT
The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.
Subject(s)
Photoreceptors, Microbial/radiation effects , Photoreceptors, Plant/radiation effects , Spectroscopy, Fourier Transform Infrared/methods , Binding Sites , Crystallography, X-Ray , Cysteine/chemistry , Flavin Mononucleotide/chemistry , Hydrogen Bonding , Models, Molecular , Photobleaching , Photochemistry , Photoreceptors, Microbial/chemistry , Photoreceptors, Plant/chemistry , Protein Conformation , Protein Domains , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/radiation effects , Subtraction TechniqueABSTRACT
The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.
Subject(s)
Bacterial Proteins/metabolism , Bacterial Proteins/radiation effects , Flavoproteins/metabolism , Flavoproteins/radiation effects , Fluorine/metabolism , Light , Photoreceptors, Microbial/metabolism , Photoreceptors, Microbial/radiation effects , Tyrosine/metabolism , Binding Sites , Color , Electron Transport , Flavin-Adenine Dinucleotide/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Protein Domains , Protons , Synechocystis/chemistryABSTRACT
The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2âO group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2âO group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of â¼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.
Subject(s)
Bacterial Proteins , Photoreceptors, Microbial , Bacterial Proteins/metabolism , Spectrum Analysis , Photoreceptors, Microbial/chemistry , Bacillus subtilis/metabolism , Flavin Mononucleotide/metabolismABSTRACT
Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.
Subject(s)
Light , Optogenetics/methods , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , HEK293 Cells , Humans , Protein Binding/radiation effects , Proteins/chemistry , Proteins/metabolismABSTRACT
A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.
Subject(s)
Optogenetics/methods , Synthetic Biology/methods , Animals , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Protein Binding , Protein Transport/physiology , Signal Transduction/physiologyABSTRACT
Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.
Subject(s)
Bacterial Proteins/metabolism , Flavins/metabolism , Flavoproteins/metabolism , Light , Photoreceptors, Microbial/metabolism , Tryptophan/analogs & derivatives , Bacterial Proteins/chemistry , Bacterial Proteins/radiation effects , Flavins/chemistry , Flavins/radiation effects , Flavoproteins/chemistry , Flavoproteins/radiation effects , Fluorescence Resonance Energy Transfer , Hydrogen Bonding/radiation effects , Molecular Conformation , Molecular Dynamics Simulation , Photoreceptors, Microbial/chemistry , Photoreceptors, Microbial/radiation effects , Tryptophan/chemistry , Tryptophan/metabolism , Tryptophan/radiation effectsABSTRACT
Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 µs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone CâO of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.
Subject(s)
Avena/chemistry , Glutamine/chemistry , Phototropins/metabolism , Protein Unfolding/radiation effects , Flavin Mononucleotide/metabolism , Hydrogen Bonding , Light , Membrane Proteins/metabolism , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Optogenetics , Phototropins/genetics , Phototropins/radiation effects , Protein Binding , Protein Conformation, alpha-Helical , Protein Domains , Protein Multimerization/radiation effectsABSTRACT
Real-time observation of structure changes associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here, we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, namely, PixD and AppABLUF; both proteins retain the red-shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins and a differential response of the two sites to chromophore excitation.
Subject(s)
Azides/chemistry , Flavoproteins/chemistry , Molecular Probes/chemistry , Phenylalanine/analogs & derivatives , Amino Acid Substitution , Amino Acids/chemistry , Flavins/chemistry , Flavoproteins/genetics , Flavoproteins/radiation effects , Hydrogen Bonding , Light , Mutation , Phenylalanine/chemistry , Protein Conformation/radiation effects , Protein Domains/radiation effects , Protein Structure, Tertiary/radiation effects , Spectrophotometry, InfraredABSTRACT
Protein/RNA clusters arise frequently in spatially regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional biochemical feedback loops.
Subject(s)
Memory, Long-Term/physiology , Organelles/chemistry , Spatial Memory/physiology , Computer Simulation , Feedback, Physiological , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Optogenetics/methods , Proteins/chemistry , RNA/analysis , Signal TransductionABSTRACT
Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.
Subject(s)
Luminescent Proteins/chemistry , Spectrophotometry, Infrared , Photochemical Processes , Protein Conformation , ProtonsABSTRACT
The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.