RESUMEN
Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.
Asunto(s)
Histonas/metabolismo , Metiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Genoma Fúngico , Código de Histonas , Histona Demetilasas/metabolismo , Histonas/química , Lisina/metabolismo , Metilación , Modelos Estadísticos , Optogenética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control subcellular localization and activity. We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic "tool kit" for the research community.
Asunto(s)
Avena/metabolismo , Fototropinas/metabolismo , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Polarización de Fluorescencia , Células HeLa , Humanos , Luz , Mutagénesis , Fototropinas/química , Fototropinas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
Cellular signal transduction is often regulated at multiple steps to achieve more complex logic or precise control of a pathway. For instance, some signaling mechanisms couple allosteric activation with localization to achieve high signal to noise. Here, we create a system for light-activated nuclear import that incorporates two levels of control. It consists of a nuclear import photoswitch, light-activated nuclear shuttle (LANS), and a protein engineered to preferentially interact with LANS in the dark, Zdk2. First, Zdk2 is tethered to a location in the cytoplasm that sequesters LANS in the dark. Second, LANS incorporates a nuclear localization signal (NLS) that is sterically blocked from binding to the nuclear import machinery in the dark. If activated with light, LANS both dissociates from its tethered location and exposes its NLS, which leads to nuclear accumulation. We demonstrate that this coupled system improves the dynamic range of LANS in mammalian cells, yeast, and Caenorhabditis elegans and provides tighter control of transcription factors that have been fused to LANS.
Asunto(s)
Optogenética/métodos , Ingeniería de Proteínas/métodos , Transporte Activo de Núcleo Celular , Animales , Caenorhabditis elegans , Células HEK293 , Células HeLa , Humanos , Luz , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL-LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Imagen Individual de Molécula/métodos , Biotinilación , Biología Computacional , Cisteína/química , Dimerización , Células HEK293 , Humanos , Lipoproteína Lipasa/química , Lipoproteína Lipasa/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Proteínas Recombinantes/química , Triglicéridos/metabolismoRESUMEN
Sactipeptides are ribosomally synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from Clostridium thermocellum ATCC 27405, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (ß/α)6-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB.
Asunto(s)
Clostridium thermocellum/enzimología , Proteínas Hierro-Azufre/metabolismo , Péptidos/metabolismo , Sulfuros/metabolismo , Secuencia de Aminoácidos , Vías Biosintéticas , Clostridium thermocellum/química , Clostridium thermocellum/metabolismo , Cristalografía por Rayos X , Proteínas Hierro-Azufre/química , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sulfuros/químicaRESUMEN
LOVTRAP is an optogenetic approach for reversible light-induced protein dissociation using protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in the dissociation constant (Kd). By reversibly sequestering proteins at mitochondria, we precisely modulated the proteins' access to the cell edge, demonstrating a naturally occurring 3-mHz cell-edge oscillation driven by interactions of Vav2, Rac1, and PI3K proteins.
Asunto(s)
Luz , Optogenética/métodos , Fosfatidilinositol 3-Quinasa/química , Fotorreceptores de Plantas , Proteínas Proto-Oncogénicas c-vav/química , Proteína de Unión al GTP rac1/química , Avena/metabolismo , Células HeLa , Humanos , Cinética , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/efectos de la radiación , Fotorreceptores de Plantas/química , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/efectos de la radiación , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/efectos de la radiación , Proteínas Recombinantes de Fusión , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/efectos de la radiaciónRESUMEN
The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular/química , Proteínas Quinasas Asociadas a Muerte Celular/metabolismo , Sitios de Unión , Proteínas Quinasas Asociadas a Muerte Celular/genética , Dimerización , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.
Asunto(s)
Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Epigénesis Genética/efectos de la radiación , Flavoproteínas/metabolismo , Histonas/metabolismo , Luz , Ubiquitinación/efectos de la radiación , Flavoproteínas/química , Flavoproteínas/genética , Señales de Exportación Nuclear , Fototropinas/química , Dominios Proteicos , Transporte de Proteínas/efectos de la radiación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.
Asunto(s)
Dimerización , Luz , Optogenética/métodos , Animales , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Criptocromos/metabolismo , Citoplasma/metabolismo , Cinética , Ratones , Mitocondrias/metabolismo , Seudópodos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Fracciones Subcelulares/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.
Asunto(s)
Núcleo Celular/metabolismo , Luz , Fototropinas/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Avena/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Embrión no Mamífero/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Señales de Localización Nuclear , Fototropinas/química , Proteínas de Plantas/química , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Degenerate codon (DC) libraries efficiently address the experimental library-size limitations of directed evolution by focusing diversity toward the positions and toward the amino acids (AAs) that are most likely to generate hits; however, manually constructing DC libraries is challenging, error prone and time consuming. This paper provides a dynamic programming solution to the task of finding the best DCs while keeping the size of the library beneath some given limit, improving on the existing integer-linear programming formulation. It then extends the algorithm to consider multiple DCs at each position, a heretofore unsolved problem, while adhering to a constraint on the number of primers needed to synthesize the library. In the two library-design problems examined here, the use of multiple DCs produces libraries that very nearly cover the set of desired AAs while still staying within the experimental size limits. Surprisingly, the algorithm is able to find near-perfect libraries where the ratio of amino-acid sequences to nucleic-acid sequences approaches 1; it effectively side-steps the degeneracy of the genetic code. Our algorithm is freely available through our web server and solves most design problems in about a second.
Asunto(s)
Algoritmos , Codón/genética , Biología Computacional/métodos , Biblioteca de Genes , Programas Informáticos , Internet , Reproducibilidad de los ResultadosRESUMEN
The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
Asunto(s)
Luz , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Multimerización de Proteína/efectos de la radiación , Transducción de Señal/efectos de la radiación , Secuencia de Aminoácidos , Avena , Técnicas de Visualización de Superficie Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas de Plantas/química , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de la radiación , Fracciones Subcelulares/metabolismoRESUMEN
When expression or crystallisation of a protein target in its wild-type full-length form proves problematic, a common strategy is to divide it into subconstructs comprising one or more domains. Rational construct design is not always successful, especially with targets for which there are few similar sequences to generate multiple sequence alignments. Even when this is possible, expression constructs may still fail to yield soluble protein, commonly expressing insolubly or at unusable yields. To address this, several new methods have been described that borrow concepts from the field of directed evolution whereby a random library is generated encompassing construct diversity; this is then screened to identify soluble constructs empirically. Here, we review progress in this area.
Asunto(s)
Automatización de Laboratorios/métodos , Biblioteca de Genes , Vectores Genéticos/química , Ensayos Analíticos de Alto Rendimiento , Proteínas/química , Clonación Molecular , Fragmentación del ADN , Desoxirribonucleasas , Escherichia coli , Expresión Génica , Genes Reporteros , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/análisis , Humanos , Mutagénesis , Proteínas/genética , Proteínas/metabolismo , SolubilidadRESUMEN
Here we present ORF-selector ESPRIT, a 9-fold enhanced version of our technology for screening incremental truncation libraries to identify soluble high yielding constructs of challenging proteins. Gene fragments are truncated at both termini to access internal domains and the resulting reading frame problem is addressed by an unbiased, intein-based open reading frame selection yielding only in-frame DNA inserts. This enriched library is then subcloned into a standard high-level expression plasmid where tens of thousands of constructs can be assayed in a two-step process using colony- and liquid-handling robots to isolate rare highly expressing clones useful for production of multi milligram quantities of purifiable proteins. The p85α protein was used to benchmark the system resulting in isolation of all known domains, either alone or in tandem. The human kinase IKK1 was then screened resulting in purification of a predicted internal domain. This strategy provides an integrated, facile route to produce soluble proteins from challenging and poorly understood target genes at quantities compatible with structural biology, screening applications and immunisation studies. The high genetic diversity that can be sampled opens the way to study more diverse systems including multisubunit complexes.
Asunto(s)
Clonación Molecular/métodos , Biblioteca de Genes , Sistemas de Lectura Abierta , Fragmentos de Péptidos/genética , Proteínas Recombinantes/genética , Automatización de Laboratorios , Fosfatidilinositol 3-Quinasa Clase Ia/biosíntesis , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Escherichia coli/genética , Vectores Genéticos , Humanos , Quinasa I-kappa B/biosíntesis , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Valores de Referencia , SolubilidadRESUMEN
Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.