SwiftLib: rapid degenerate-codon-library optimization through dynamic programming

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

Light-induced nuclear export reveals rapid dynamics of epigenetic modifications

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

Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide

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 co..

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Photoactivatable proteins are powerful tools for studying biological processes. Light-induced dimers are especially useful because they can be turned on and off with high spatial and temporal resolution in living systems, allowing for control of protein localization and activity. Here, we develop and apply methods for identifying mutations that improve the effectiveness of a light-induced dimer. The engineered switch is modular, can be used in most organisms, has more than 50-fold change in binding affinity upon light stimulation, and can be used to initiate signaling pathways in a specific region of a cell

LOVTRAP: an optogenetic system for photoinduced protein dissociation

Here we introduce LOVTRAP, an optogenetic approach for reversible, light-induced protein dissociation. LOVTRAP is based on protein A fragments that bind to the LOV domain only in the dark, with tunable kinetics and a >150-fold change in 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

Caractérisation Structurale des Kinase Humaines par le Criblage de Bibliothèque des Constructions

Structural characterization of proteins is often hindered by insufficient amounts of soluble protein. A common approach is to isolate its separate domains, classically done by time-consuming iterations of design, generation and testing of constructs. An alternative approach is to generate a random library of all possible constructs by enzymatic DNA truncation and test them in one experiment for expression and solubility. In this work, the novel, directed evolution-type method Expression of Soluble Proteins by Random Incremental Truncations (ESPRIT) was used to explore the definition of protein domains. The biological focus was a set of multidomain protein kinases that has previously resisted soluble over-expression and structural characterisation. First, improvements in the method were developed to improve the quality and efficiency of the truncation libraries leading to better coverage of the diversity. Then, its application on a more characterised protein, DAPK1, demonstrated precise domain definition. Small variations of the constructs resulted in significantly different thermal stability and crystal packing, thus providing a means to resolve structures with alternative conformations. The method was also used for the identification of soluble variants of the complex between DAPK1 and its partner calmodulin where the minimal interaction region was shorter than previously reported. The application of the method to a series of difficult-to-express protein kinases resulted in rapid identification of incompatibility with E. coli over-expression for some of the targets. While attempts to rescue the low solubility the IKKb kinase catalytic domain were unsuccessful, additional screens on this protein identified soluble and regulatory domains that showed some functionality in vivo. Finally, it was demonstrated that constructs of the PI3Kb p110b catalytic domain with residual solubility in E. coli, identified from screening, could be expressed in insect cells at higher yields in soluble form.Le manque de protéine soluble est souvent un obstacle à la caractérisation structurale des protéines. Une approche courante pour surmonter cela consiste à isoler des domaines protéiques en utilisant des méthodes itératives de création et analyse de constructions. Autrement des banques d'ADN, contenant toutes les constructions possibles d'une même protéine, peuvent être crée par troncation enzymatique et testée à la fois pour l'expression et la solubilité de celle-ci. Dans ce projet, la nouvelle méthode d'Expression des Protéines Solubles par Troncation Aléatoire Incrémentielle (ESPRIT) a été utilisée pour explorer la définition des domaines protéiques. Des protéines kinases multidomaines qui avaient échoué surexpression solubles ont été choisies pour leur intérêt biologique. La méthode a d'abord été optimisée pour améliorer la qualité et l'efficacité des banques permettant un meilleur traitement de la diversité générée. Elle a ensuite été appliquée à une protéine kinase modèle DAPK1, à partir de laquelle une définition précise de domaine a été démontrée. Le criblage des constructions a ainsi permis l'identification de protéines plus stables, et cristallisation des constructions dans les conformations alternatives. La méthode a aussi été appliquée pour identifier des variants solubles du complexe DAPK1 et son partenaire la calmoduline, permettant de mettre en évidence un domaine d'interaction minimal, plus petit que celui décrit auparavant. Alors que plusieurs tentatives pour obtenir le domaine catalytique kinase de IKKb avaient échoué, des criblages additionnels sur cette protéine avec cette méthode ont permis d'identifier des domaines de régulation solubles et fonctionnels in vivo. Enfin, il a été démontré que des constructions du domaine catalytique de p110b, faiblement exprimées dans E.coli, pouvaient être exprimées solubles dans des cellules d'insectes avec des rendements plus importants

Caractérisation structurale de kinases humaines à partir d'un crible de banques de polypeptides

Obtenir des quantités suffisantes de protéines solubles est souvent un obstacle à la caractérisation structurale des protéines. Une approche pour surmonter cela est d'isoler des domaines protéiques en utilisant des méthodes itératives de création et d'analyse de constructions. Un moyen d'y parvenir consiste à créer, par troncation enzymatique, des banques d'ADN, contenant toutes les constructions possibles d'une même protéine, et à tester à la fois l'expression et la solubilité de celles-ci. Durant la réalisation de cette thèse, une nouvelle méthode, appellée ESPRIT pour Expression of Soluble Proteins by Random Incremental Truncation, a été utilisée pour définir des domaines protéiques. Plusieurs protéines kinases avec des domaines multiples, essentiels aux fonctions cellulaires et dont la production sous une forme soluble avait échoué auparavant, ont été analysées par cette nouvelle technologie. Cette méthode a d'abord été optimisée pour améliorer la qualité et l'efficacité des banques permettant un meilleur traitement de la diversité des constructions générées. Elle a ensuite été appliquée à une protéine kinase modèle DAPK1, pour laquelle une définition précise de domaine a été démontrée. Des petites variations de constructions ont donné des stabilités thermiques et des assemblages cristallins différents permettant ainsi la cristallisation dans des conformations différentes. La méthode a aussi été appliquée pour identifier des variants solubles du complexe DAPK1 et son partenaire la calmoduline, permettant de mettre en évidence un domaine d'intéraction minimal, plus petit que celui décrit auparavant. Alors que plusieurs tentatives pour obtenir le domaine catalytique kinase de IKKb avaient échoué, des criblages additionnels sur cette protéine avec cette méthode ont permis d'identifier des domaines de régulation solubles et fonctionnels in vivo. Enfin, il a été démontré que des constructions du domaine catalytique de p110b, faiblement exprimées dans E.coli, pouvaient être exprimées solubles dans des cellules d'insectes avec des rendements plus importants.Structural characterisation of proteins is often hindered by insufficient amounts of soluble material. A common approach addressing this problem is to isolate their separate domains, classically done by time-consuming iterations of design, generation and testing of constructs. An alternative approach is to generate a random library of all possible constructs by enzymatic DNA truncation and test them in one experiment for expression and solubility. In this work, the novel, directed evolution-type method Expression of Soluble Proteins by Random Incremental Truncations (ESPRIT) was used to explore the definition of proteins domains. The biological focus was a set of multidomain protein kinases that has previously resisted soluble over-expression and structural characterisation. First, improvements in the method were developed to improve the quality and efficiency of the truncation librairies leading to better coverage of the diversity. Then, its application on a more characterised protein, DAPK1, demonstrated precise domain definition. Small variations of the constructs resulted in significantly different thermal stability and crystal packing, thus providing a means to resolve a structure with an alternative conformation. The method was also used for the identification of soluble variants of the complex between DAPK1 and its partner calmodulin where the minimal interaction region was shorter than previously report. The application of the method to a series of difficult-to-express protein kinases resulted in rapid identification of incompatibility with E. coli over-expression for some of the targets. While attempts to rescue the low solubility the IKKb kinase catalytic domain were unsuccessful, additional screens on this protein identified soluble and regulatory domains that showed some functionality in vivo. Finally, it was demonstrated that constructs of the PI3Kb p110b catalytic domain with residual solubility in E. coli, identified from screening, could be expressed in insect cells at higher yields in soluble form.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Correlating <i>in Vitro</i> and <i>in Vivo</i> Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide

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 <i>in vivo</i> 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 <i>in vivo</i> 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 <i>in vivo</i> 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

We FRET so You Don’t Have To: New Models of the Lipoprotein Lipase Dimer

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

An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and -independent dynamics of H3K36 methylation.

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