RESUMEN
Azonium ions formed by the protonation of tetra-ortho-methoxy-substituted aminoazobenzenes photoisomerize with red light under physiological conditions. This property makes them attractive as molecular tools for the photocontrol of physiological processes, for example, in photopharmacology. However, a mechanistic understanding of the photoisomerization process and subsequent thermal relaxation is necessary for the rational application of these compounds as well as for guiding the design of derivatives with improved properties. Using a combination of sub-ps/ns transient absorption measurements and quantum chemical calculations, we show that the absorption of a photon by the protonated E-H+ form of the photoswitch causes rapid (ps) isomerization to the protonated Z-H+ form, which can also absorb red light. Proton transfer to solvent then occurs on a microsecond time scale, leading to an equilibrium between Z and Z-H+ species, the position of which depends on the solution pH. Whereas thermal isomerization of the neutral Z form to the neutral E form is slow (â¼0.001 s-1), thermal isomerization of Z-H+ to E-H+ is rapid (â¼100 s-1), so the solution pH also governs the rate at which E/E-H+ concentrations are restored after a light pulse. This analysis provides the first complete mechanistic picture that explains the observed intricate photoswitching behavior of azonium ions at a range of pH values. It further suggests features of azonium ions that could be targeted for improvement to enhance the applicability of these compounds for the photocontrol of biomolecules.
RESUMEN
The acylhydrazone unit is well represented in screening databases used to find ligands for biological targets, and numerous bioactive acylhydrazones have been reported. However, potential E/Z isomerization of the C=N bond in these compounds is rarely examined when bioactivity is assayed. Here we analysed two ortho-hydroxylated acylhydrazones discovered in a virtual drug screen for modulators of N-methyl-D-aspartate receptors and other bioactive hydroxylated acylhydrazones with structurally defined targets reported in the Protein Data Bank. We found that ionized forms of these compounds, which are populated under laboratory conditions, photoisomerize readily and the isomeric forms have markedly different bioactivity. Furthermore, we show that glutathione, a tripeptide involved with cellular redox balance, catalyses dynamic EâZ isomerization of acylhydrazones. The ratio of E to Z isomers in cells is determined by the relative stabilities of the isomers regardless of which isomer was applied. We conclude that E/Z isomerization may be a common feature of the bioactivity observed with acylhydrazones and should be routinely analysed.
Asunto(s)
Compuestos de Sulfhidrilo , Isomerismo , Bases de Datos de ProteínasRESUMEN
Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.
Asunto(s)
Cianobacterias , Animales , Cianobacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Luz , Optogenética , Mamíferos/metabolismoRESUMEN
Thiol-reactive reagents designed for the chemical modification of proteins cannot, in general, be used directly for the modification of intracellular targets because the presence of millimolar concentrations of glutathione inside cells effectively outcompetes reaction with target thiols. Here we report an equilibrium, entropic strategy for achieving target selectivity using a cyanoacrylate-based thiol-reactive cross-linker (BCNA) with two reactive sites. This compound exhibits â³200-fold selectivity for reaction with target peptides and proteins containing appropriately spaced pairs of thiols, versus reaction with mono-thiols. Photo-isomerization of the azobenzene moiety of the cross-linker can be used to affect the conformation of the target peptide or protein. This approach suggests a general strategy for the chemical modification of intracellular peptide and protein targets.
Asunto(s)
Compuestos Azo , Proteínas , Reactivos de Enlaces Cruzados/química , Compuestos Azo/química , Péptidos/química , Compuestos de Sulfhidrilo/químicaRESUMEN
Cyanobacteriochromes (CBCRs) are photoreceptors consisting of single or tandem GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domains that bind bilin chromophores. Canonical red/green CBCR GAF domains are a well-characterized subgroup of the expanded red/green CBCR GAF domain family that binds phycocyanobilin (PCB) and converts between a thermally stable red-absorbing Pr state and a green-absorbing Pg state. The rate of thermal reversion from Pg to Pr varies widely among canonical red/green CBCR GAF domains, with half-lives ranging from days to seconds. Since the thermal reversion rate is an important parameter for the application of CBCR GAF domains as optogenetic tools, the molecular factors controlling the thermal reversion rate are of particular interest. Here, we report that point mutations in a well-conserved W(S/G)GE motif alter reversion rates in canonical red/green CBCR GAF domains in a predictable manner. Specifically, S-to-G mutations enhance thermal reversion rates, while the reverse, G-to-S mutations slow thermal reversion. Despite the distance (>10 Å) of the mutation site from the chromophore, molecular dynamics simulations and nuclear magnetic resonance (NMR) analyses suggest that the presence of a glycine residue allows the formation of a water bridge that alters the conformational dynamics of chromophore-interacting residues, leading to enhanced Pg to Pr thermal reversion.
Asunto(s)
Fotorreceptores Microbianos , Fitocromo , Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/química , Pigmentos Biliares , Luz , Conformación Molecular , Mutación , Fotorreceptores Microbianos/química , Fitocromo/químicaRESUMEN
Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.
Asunto(s)
Evolución Molecular Dirigida , Optogenética/métodos , Luz , Levaduras/metabolismoRESUMEN
Photo-control of affinity reagents offers a general approach for high-resolution spatiotemporal control of diverse molecular processes. In an effort to develop general design principles for a photo-controlled affinity reagent, we took a structure-based approach to the design of a photoswitchable Z-domain, among the simplest of affinity reagent scaffolds. A chimera, designated Z-PYP, of photoactive yellow protein (PYP) and the Z-domain, was designed based on the concept of mutually exclusive folding. NMR analysis indicated that, in the dark, the PYP domain of the chimera was folded, and the Z-domain was unfolded. Blue light caused loss of structure in PYP and a two- to sixfold change in the apparent affinity of Z-PYP for its target as determined using size exclusion chromatography, UV-Vis based assays, and enyzme-linked immunosorbent assay (ELISA). A thermodynamic model indicated that mutations to decrease Z-domain folding energy would alter target affinity without loss of switching. This prediction was confirmed experimentally with a double alanine mutant in helix 3 of the Z-domain of the chimera (Z-PYP-AA) showing >30-fold lower dark-state binding and no loss in switching. The effect of decreased dark-state binding affinity was tested in a two-hybrid transcriptional control format and enabled pronounced light/dark differences in yeast growth in vivo. Finally, the design was transferable to the αZ-Taq affibody enabling tunable light-dependent binding both in vitro and in vivo to the Z-Taq target. This system thus provides a framework for the focused development of light switchable affibodies for a range of targets.
Asunto(s)
Anticuerpos/química , Proteínas Bacterianas/química , Optogenética/métodos , Fotorreceptores Microbianos/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Anticuerpos/metabolismo , Afinidad de Anticuerpos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Luz , Procesos Fotoquímicos , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Although widely used in the detection and characterization of protein-protein interactions, Y2H screening has been under-used for the engineering of new optogenetic tools or the improvement of existing tools. Here we explore the feasibility of using Y2H selection and screening to evaluate libraries of photoswitchable protein-protein interactions. We targeted the interaction between circularly permuted photoactive yellow protein (cPYP) and its binding partner binder of PYP dark-state (BoPD) by mutating a set of four surface residues of cPYP that contribute to the binding interface. A library of ~10,000 variants was expressed in yeast together with BoPD in a Y2H format. An initial selection for the cPYP/BoPD interaction was performed using a range of concentrations of the cPYP chromophore. As expected, the majority (>90% of cPYP variants) no longer bound to BoPD. Replica plating was then used to evaluate the photoswitchability of the surviving clones. Photoswitchable cPYP variants with BoPD affinities equal to, or higher than, native cPYP were recovered in addition to variants with altered photocycles and binders that interacted with BoPD as apo-proteins. Y2H results reflected protein-protein interaction affinity, expression, photoswitchability, and chromophore uptake, and correlated well with results obtained both in vitro and in mammalian cells. Thus, by systematic variation of selection parameters, Y2H screens can be effectively used to generate new optogenetic tools for controlling protein-protein interactions for use in diverse settings.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fotorreceptores Microbianos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estudios de Factibilidad , Biblioteca de Genes , Humanos , Modelos Moleculares , Optogenética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Unión Proteica , Proteínas/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") . Previously, we and others showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram. In these studies, though, CREB function was artificially increased for several days before training. Precisely when increased CREB function is important for allocation remains an unanswered question. Here, we took advantage of a novel optogenetic tool (opto-DN-CREB) to gain spatial and temporal control of CREB function in freely behaving mice. We found increasing CREB function in a small, random population of LA principal neurons in the minutes, but not 24 h, before training was sufficient to enhance memory, likely because these neurons were preferentially allocated to the underlying engram. However, similarly increasing CREB activity in a small population of random LA neurons immediately after training disrupted subsequent memory retrieval, likely by disrupting the precise spatial and temporal patterns of offline post-training neuronal activity and/or function required for consolidation. These findings reveal the importance of the timing of CREB activity in regulating allocation and subsequent memory retrieval, and further, highlight the potential of optogenetic approaches to control protein function with temporal specificity in behaving animals.
Asunto(s)
Complejo Nuclear Basolateral , Optogenética , Animales , Miedo , Memoria , Ratones , NeuronasRESUMEN
The photo-control of protein activity can often be achieved via the photo-control of protein structure. Intramolecular cross-linkers that change length upon photoisomerization provide a means to photo-control protein structure by linking to pairs of Cys residues in a protein sequence. In this protocol, we describe general methods for introducing intramolecular cross-linkers, both UV light switchable and red-light switchable, under either denaturing or native conditions.
Asunto(s)
Compuestos Azo/química , Reactivos de Enlaces Cruzados/química , Cisteína/química , Proteínas/química , Secuencia de Aminoácidos , Compuestos Azo/síntesis química , Técnicas de Química Sintética/métodos , Cromatografía Líquida de Alta Presión/métodos , Reactivos de Enlaces Cruzados/síntesis química , Cisteína/síntesis química , Luz , Modelos Moleculares , Procesos Fotoquímicos , Conformación Proteica , Pliegue de Proteína , Proteínas/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrofotometría Ultravioleta/métodos , Rayos UltravioletaRESUMEN
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce â¼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fotorreceptores Microbianos/metabolismo , Propionatos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Amoníaco-Liasas/metabolismo , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Coenzima A Ligasas/metabolismo , Ácidos Cumáricos , Escherichia coli/genética , Fluorescencia , Halorhodospira halophila/química , Cinética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Mutación Puntual , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development, and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis noninvasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, AsLOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photoactivated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate human eIF4E-dependent translation initiation in a mechanistically defined manner.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Optogenética/métodos , Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas/genética , Saccharomyces cerevisiae/genética , Regulación hacia Abajo/genética , Humanos , Unión Proteica/genéticaRESUMEN
We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to ß-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for ß-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Mapas de Interacción de Proteínas/genética , Ingeniería de Proteínas/métodos , beta-Lactamasas/genéticaRESUMEN
Photo-controlled affinity reagents seek to provide modular spatiotemporal control of bioactivity by conferring photo-switchability of function on an affinity reagent scaffold. Here we used Rosetta-based computational methods to screen for sites on the Fynomer affinity reagent structure for attachment of photoswitchable cross-linkers. Both established UV-based cross-linkers (azobenzene-iodoacetamide (IAC)) and an azonium-based efficient red light switchable cross-linker, piperazino-tetra-ortho-methoxy azobenzene (PIP), were then tested experimentally. Several sites compatible with Fynomer function were identified, including sites showing rapid (<10s) red light (633 nm) modulation of function. While a range of overall target binding affinities were observed, the degree of photo-switchability of Fynomer function was generally small (<2-fold). Computational models suggest that local flexibility limits the degree of switching seen in these designs.
RESUMEN
Aminoazobenzene derivatives with four ortho substituents with respect to the N-N double bond are a relatively unexplored class of azo compounds that show promise for use as photoswitches in biology. Tetra-ortho-methoxy-substituted aminoazobenzene compounds in particular can form azonium ions under physiological conditions and exhibit red-light photoswitching. Here, we report the synthesis and characterization of two bis(4-amino-2-bromo-6-methoxy)azobenzene derivatives. These compounds form red-light-absorbing azonium ions, but only under very acidic conditions (pH < 1). While the low pK a makes the azonium form unsuitable, the neutral versions of these compounds undergo trans-to-cis photoisomerization with blue-green light and exhibit slow (τ1/2 ≈ 10 min) thermal reversion and so may find applications under physiological conditions.
RESUMEN
Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control-precise spatial and temporal resolution-are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often suboptimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2, a well-characterized protein. We show that these novel light-switchable protein-protein interactions can function in living cells to control subcellular localization processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Luz , Fotorreceptores Microbianos/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Espectroscopía de Resonancia Magnética , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Duplication of a single ß-strand that forms part of a ß-sheet in photoactive yellow protein (PYP) was found to produce two approximately isoenergetic protein conformations, in which either the first or the second copy of the duplicated ß-strand participates in the ß-sheet. Whereas one conformation (big-loop) is more stable at equilibrium in the dark, the other conformation (long-tail) is populated after recovery from blue light irradiation. By appending a recognition motif (E-helix) to the C-terminus of the protein, we show that ß-strand duplication, and the resulting possibility of ß-strand slippage, can lead to a new switchable protein-protein interaction. We suggest that ß-strand duplication may be a general means of introducing two-state switching activity into protein structures.
Asunto(s)
Proteínas Bacterianas/química , Halorhodospira halophila/química , Fotorreceptores Microbianos/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Halorhodospira halophila/genética , Luz , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fotorreceptores Microbianos/genética , Conformación Proteica , Conformación Proteica en Lámina betaRESUMEN
There is growing interest in designing spatiotemporal control over enzyme activities using noninvasive stimuli such as light. Here, we describe a structure-based, computation-guided predictive method for reversibly controlling enzyme activity using covalently attached photoresponsive azobenzene groups. Applying the method to the therapeutically useful enzyme yeast cytosine deaminase, we obtained a â¼3-fold change in enzyme activity by the photocontrolled modulation of the enzyme's active site lid structure, while fully maintaining thermostability. Multiple cycles of switching, controllable in real time, are possible. The predictiveness of the method is demonstrated by the construction of a variant that does not photoswitch as expected from computational modeling. Our design approach opens new avenues for optically controlling enzyme function. The designed photocontrolled cytosine deaminases may also aid in improving chemotherapy approaches that utilize this enzyme.
Asunto(s)
Compuestos Azo/química , Citosina Desaminasa/química , Citosina Desaminasa/efectos de la radiación , Procesos Fotoquímicos , Compuestos Azo/metabolismo , Citosina Desaminasa/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/enzimologíaRESUMEN
Biological tissue exhibits an absorbance minimum in the near-infrared between 700 and 900 nm that permits deep penetration of light. Molecules that undergo photoisomerization in this bio-optical window are highly desirable as core structures for the development of photopharmaceuticals and as components of chemical-biological tools. We report the systematic design, synthesis, and testing of an azobenzene derivative tailored to undergo single-photon photoswitching with near-infrared light under physiological conditions. A fused dioxane ring and a methoxy substituent were used to place oxygen atoms in all four ortho positions, as well as two meta positions, relative to the azobenzene NâN double bond. This substitution pattern, together with a para pyrrolidine group, raises the pKa of the molecule so that it is protonated at physiological pH and absorbs at wavelengths >700 nm. This azobenzene derivative, termed DOM-azo, is stable for months in neutral aqueous solutions, undergoes trans-to-cis photoswitching with 720 nm light, and thermally reverts to the stable trans isomer with a half-life near 1 s.
Asunto(s)
Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Rayos Infrarrojos , Procesos Fotoquímicos/efectos de la radiación , Concentración de Iones de Hidrógeno , Isomerismo , Protones , Pirrolidinas/químicaRESUMEN
Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field.