ABSTRACT
Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains. Use of the Aureochrome1a light-oxygen-voltage domain delivered control of dimerization with light. Illumination reduced the concentration needed to achieve 50% cleavage of the homing target site by 6-fold when compared to the dark state, resulting in an up to 9-fold difference in final yields between cleavage products. I-TevI nucleases with and without a native I-TevI zinc finger motif displayed different nuclease activity and sequence preference impacting the promiscuity of the nuclease domain. By harnessing an alternative DNA binding domain, target preference was reprogrammed only when the nuclease lacked the I-TevI zinc finger motif. This work establishes a first-generation photoresponsive platform for spatiotemporal activation of DNA cleavage.
Subject(s)
Endodeoxyribonucleases , Endonucleases , Endonucleases/genetics , Endonucleases/metabolism , Base Sequence , Endodeoxyribonucleases/genetics , DNA Cleavage , DNA/metabolismABSTRACT
Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.
Subject(s)
Antineoplastic Agents , Cell-Penetrating Peptides , Inteins , Protein Splicing , Recombinant ProteinsABSTRACT
A nuclear localisation sequence (NLS) peptide, PAAKRVKLD, derived from the human c-Myc regulator gene, has been functionalised with a long wavelength (λ ex = 550 nm; λ em = 677 nm) cyclometalated organometallic iridium(iii) complex to give the conjugate Ir-CMYC. Confocal fluorescence microscopy studies on human fibroblast cells imaged after 18-24 h incubation show that Ir-CMYC concentrations of 80-100 µM promote good cell uptake and nuclear localisation, which was confirmed though co-localisation studies using Hoechst 33342. In comparison, a structurally related, photophysically analogous iridium(iii) complex lacking the peptide sequence, Ir-PYR, showed very different biological behaviour, with no evidence of nuclear, lysosomal or autophagic vesicle localisation and significantly increased toxicity to the cells at concentrations >10 µM that induced mitochondrial dysfunction. Supporting UV-visible and circular dichroism spectroscopic studies show that Ir-PYR and Ir-CMYC display similarly low affinities for DNA (ca. 103 M-1), consistent with electrostatic binding. Therefore the translocation and nuclear uptake properties of Ir-CMYC are attributed to the presence of the PAAKRVKLD nuclear localisation sequence in this complex.
ABSTRACT
Non-natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large-scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)-FDP and (2Z,6Z)-FDP, were isolated in greater than 80 % yields. The synthesis of 14,15-dimethyl-FDP, 12-methyl-FDP, 12-hydroxy-FDP, homo-FDP, and 15-methyl-FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)-14,15-dimethylgermacreneâ D and (S)-12-methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non-natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.
Subject(s)
Dimethylallyltranstransferase/metabolism , Terpenes/chemistry , Terpenes/chemical synthesis , Chemistry Techniques, Synthetic , Phosphorylation , Polyisoprenyl Phosphates/chemistry , Sesquiterpenes/chemistryABSTRACT
Light-oxygen-voltage (LOV) domains are increasingly used to engineer photoresponsive biological systems. While the photochemical cycle is well documented, the allosteric mechanism by which formation of a cysteinyl-flavin adduct leads to activation is unclear. Via replacement of flavin mononucleotide (FMN) with 5-deazaflavin mononucleotide (5dFMN) in the Aureochrome1a (Au1a) transcription factor from Ochromonas danica, a thermally stable cysteinyl-5dFMN adduct was generated. High-resolution crystal structures (<2 Å) under different illumination conditions with either FMN or 5dFMN chromophores reveal three conformations of the highly conserved glutamine 293. An allosteric hydrogen bond network linking the chromophore via Gln293 to the auxiliary A'α helix is observed. With FMN, a "flip" of the Gln293 side chain occurs between dark and lit states. 5dFMN cannot hydrogen bond through the C5 position and proved to be unable to support Au1a domain dimerization. Under blue light, the Gln293 side chain instead "swings" away in a conformation distal to the chromophore and not previously observed in existing LOV domain structures. Together, the multiple side chain conformations of Gln293 and functional analysis of 5dFMN provide new insight into the structural requirements for LOV domain activation.
Subject(s)
Algal Proteins/chemistry , Flavins/chemistry , Ribonucleotides/chemistry , Transcription Factors/chemistry , Algal Proteins/radiation effects , Cysteine/chemistry , Flavin Mononucleotide/chemistry , Glutamine/chemistry , Light , Ochromonas/chemistry , Protein Conformation/radiation effects , Protein Domains/radiation effects , Transcription Factors/radiation effectsABSTRACT
The last few years have witnessed significant advances in the use of light as a stimulus to control biomolecular interactions. Great efforts have been devoted to the development of genetically encoded optobiological and small photochromic switches. Newly discovered small molecules now allow researchers to build molecular systems that are sensitive to a wider range of wavelengths of light than ever before with improved switching fidelities and increased lifetimes of the photoactivated states. Because these molecules are relatively small and adopt predictable conformations they are well suited as tools to interrogate cellular function in a spatially and temporally contolled fashion and for applications in photopharmacology.
Subject(s)
Azo Compounds/chemistry , Light , Peptide Fragments/chemistry , Protein Conformation/radiation effects , Transferrin/chemistry , HeLa Cells , Humans , Peptide Fragments/radiation effects , Transferrin/radiation effectsABSTRACT
LOV domains act as biomolecular sensors for light, oxygen or the environment's redox potential. Conformational changes upon the formation of a covalent cysteinyl flavin adduct are propagated through hydrogen-bonding networks in the core of designed hybrid phototropin LOV2 domains that incorporate the Bcl homology regionâ 3 (BH3) of the key pro-apoptotic protein BH3-interacting-domain death agonist (BID). The resulting change in conformation of a flanking amphiphilic α-helix creates a light-dependent optogenetic tool for the modulation of interactions with the anti-apoptotic B-cell leukaemia-2 (Bcl-2) family member Bcl-xL .
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Bacterial Proteins/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Amino Acid Sequence , Fluorescence Polarization , Photochemical Processes , Protein Conformation , Sequence Analysis, ProteinABSTRACT
Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein-protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway.
Subject(s)
Cytochromes c/metabolism , Peptide Fragments/pharmacology , Proto-Oncogene Proteins/pharmacology , Alkylation/drug effects , Amino Acid Sequence , Azo Compounds/chemistry , Azo Compounds/pharmacology , Cell Line, Tumor , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Peptide Fragments/chemistry , Protein Structure, Secondary , Proto-Oncogene Proteins/chemistryABSTRACT
Here we report the (1)H, (13)C and (15)N resonance assignments of free Bcl-x(L) and of Bcl-x(L) in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding.
Subject(s)
Light , Nuclear Magnetic Resonance, Biomolecular , Peptides/metabolism , Protons , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-X Protein/chemistry , bcl-X Protein/metabolism , Amino Acid Sequence , Carbon Isotopes , Ligands , Nitrogen Isotopes , Peptides/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x(L). This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states.
Subject(s)
Peptides/chemistry , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/metabolism , Amino Acid Sequence , Apoptosis , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptides/metabolismABSTRACT
Positively constrained: the first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting α-helix imposes constraints on the choice of azobenzene-derived crosslinker.
Subject(s)
Peptides/chemistry , Peptidomimetics/radiation effects , Photochemical Processes , Proteins/chemistry , RNA/chemistry , Azo Compounds/chemistry , Binding Sites , Cross-Linking Reagents/chemical synthesis , Cross-Linking Reagents/chemistry , Models, Molecular , Molecular Dynamics Simulation , Peptides/chemical synthesisABSTRACT
In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.
Subject(s)
Peptides/chemical synthesis , Peptides/pharmacokinetics , Protein Engineering/methods , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/chemistry , Amino Acid Sequence , Apoptosis , Cell Line, Tumor , Cell Membrane Permeability , Cyanogen Bromide/chemistry , Cytochromes c/metabolism , Humans , Molecular Sequence Data , Oligopeptides/chemistry , Peptide Fragments/chemistry , Proto-Oncogene Proteins/chemistry , Recombinant Fusion Proteins/genetics , bcl-2 Homologous Antagonist-Killer Protein/chemistryABSTRACT
This review article covers recent developments in the use of enzyme-catalyzed reactions to control molecular self-assembly (SA), an area that merges the advantages of biocatalysis with soft materials self-assembly. This approach is attractive because it combines biological (chemo-, regio-, and enantio-) selectivity with the versatility of bottom up nanofabrication through dynamic SA. We define enzyme-assisted SA (e-SA) as the production of molecular building blocks from nonassembling precursors via enzymatic catalysis, where molecular building blocks form ordered structures via noncovalent interactions. The molecular design of SA precursors is discussed in terms of three key components related to (i) enzyme recognition, (ii) molecular switching mechanisms, and (iii) supramolecular interactions that underpin SA. This is followed by a discussion of a number of unique features of these systems, including spatiotemporal control of nucleation and structure growth, the possibility of controlling mechanical properties and the defect correcting and component selecting capabilities of systems that operate under thermodynamic control. Applications in biomedicine (biosensing, controlled release, matrices for wound healing, controlling cell fate by gelation) and bio(nano)technology (biocatalysts immobilization, nanofabrication, templating, and intracellular imaging) are discussed. Overall, e-SA allows for unprecedented control over SA processes and provides a step forward toward production of nanostructures of higher complexity and with fewer defects as desired for next generation nanomaterials.
Subject(s)
Biocatalysis , Peptides/chemistry , Enzymes/chemistry , Enzymes/metabolism , Molecular Structure , Peptide Library , Peptides/metabolism , Protein Conformation , ThermodynamicsABSTRACT
Vesicles (liposomes) have been shown to be excellent vehicles for drug delivery, yet assemblies of vesicles (vesicle aggregates) have been used infrequently in this context. However vesicle assemblies have useful properties not available to individual vesicles; their size can cause localisation in specific tissues and they can incorporate more functionality than is possible with individual vesicles. This article reviews progress on controlling the properties of vesicle assemblies in vitro, applications of vesicle assemblies in vivo, and our recent creation of magnetic nanoparticle-vesicle assemblies. The latter assemblies contain vesicles crosslinked by coated Fe3O4 nanoparticles and this inclusion of magnetic functionality makes them magnetically responsive, potentially allowing magnetically-induced contents release. This article describes further studies on the in vitro formation of these magnetic nanoparticle-vesicle assemblies, including the effect of changing magnetic nanoparticle concentration, pH, adhesive lipid structure and bilayer composition. These investigations have led to the development of thermally-sensitive magnetic nanoparticle-vesicle assemblies that release encapsulated methotrexate on warming.
Subject(s)
Drug Delivery Systems/methods , Liposomes/chemistry , Nanoparticles/chemistry , Ferric Compounds , Hydrogen-Ion Concentration , Lipids/chemistry , Magnetics , Methotrexate/chemistry , Nanoparticles/ultrastructure , Particle Size , Phase Transition , TemperatureABSTRACT
Magnetic nanoparticle-vesicle assemblies embedded within a hydrogel extravesicular matrix have been shown to release their contents in response to a remote magnetic trigger.
Subject(s)
Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Magnetics , Microscopy, Electron, Scanning , Molecular StructureABSTRACT
We report on enzyme-responsive hydrogel particles for the controlled release of proteins. Amino-functionalised poly(ethylene glycol acrylamide) (PEGA) hydrogel particles were functionalised with peptide actuators that cause charge-induced swelling and payload release when triggered enzymatically. Peptide-based actuators were designed to match the specificity of the target enzyme, while also matching the charge properties of the to-be released protein payload, thereby uniquely allowing for tuneable release profiles. Fluorescently labelled albumin and avidin, proteins of similar size but opposite charge, were released at a rate that was governed by the peptide actuator linked to the polymer carrier, offering a highly controlled release mechanism. Release profiles were analysed using a combination of fluorescence spectroscopy of the solution and two-photon fluorescence microscopy to analyse enzymatically triggered molecular events within hydrogel particles during the initial stages of release.
Subject(s)
Ferrosoferric Oxide/chemistry , Liposomes/chemistry , Magnetics , Membrane Proteins/chemistry , Nanoparticles/chemistry , Catechols/chemistry , Cholesterol/chemistry , Copper/chemistry , Dimyristoylphosphatidylcholine/chemistry , Dopamine/chemistry , Edetic Acid/chemistry , Ferric Compounds/chemistry , Histidine/analogs & derivatives , Histidine/chemistry , Microscopy, Electron, Transmission , Nanotechnology/methods , Nephelometry and TurbidimetryABSTRACT
In an effort to improve the stability of our tissue-mimetic vesicle aggregates, we have investigated how increasing the valency of our multivalent crosslinking ligand, poly-l-histidine, affected both the extent of vesicle aggregation and the affinity of the multivalent ligand for the synthetic receptor Cu(1) embedded in the vesicle membranes. Although increasing ligand valency gave the anticipated increase in the size of the vesicle aggregates, isothermal calorimetric studies did not show the expected increase in the valence-corrected binding constant for the embedded receptors. To explain both observations, we have developed a simple new binding model that encompasses both multivalent binding to receptors on a single vesicle surface (intramembrane binding) and vesicle crosslinking (intermembrane binding).
Subject(s)
Cross-Linking Reagents/chemistry , Calorimetry , Histidine/chemistry , Hydrogen/chemistry , Ligands , Lipids/chemistry , Models, Chemical , Molecular Structure , Solutions , Surface PropertiesABSTRACT
As part of our studies into how the localization of cell adhesion molecules into lipid rafts may affect cell adhesion, we developed Cu(1), a synthetic copper(iminodiacetate)-capped receptor able to phase separate from fluid phospholipid bilayers. The extent to which Cu(1) clustered into adhesive patches on the surface of vesicles could be controlled by changing vesicle composition. Extensive receptor phase separation significantly enhanced vesicle-vesicle adhesion; only vesicles with adhesive patches (blue fluorescence) adhered to their conjugate histidine-coated vesicles (red fluorescence) to form large vesicle aggregates (shown).
Subject(s)
Cell Adhesion , Receptors, Cell Surface/physiology , Microscopy, FluorescenceABSTRACT
This article explores recent advances in the design and engineering of materials wholly or principally constructed from peptides. We focus on materials that are able to respond to changes in their environment (pH, ionic strength, temperature, light, oxidation/reduction state, presence of small molecules or the catalytic activity of enzymes) by altering their macromolecular structure. Such peptide-based responsive biomaterials have exciting prospects for a variety of biomedical and bionanotechnology applications in drug delivery, bio-sensing and regenerative medicine.