The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

Highly tunable bimane-based fluorescent probes: design, synthesis, and application as a selective amyloid binding dye.

Small molecule fluorescent probes are indispensable tools for a broad range of biological applications. Despite many probes being available, there is still a need for probes where photophysical properties and biological selectivity can be tuned as desired. Here, we report the rational design and synthesis of a palette of fluorescent probes based on the underexplored bimane scaffold. The newly developed probes with varied electronic properties show tunable absorption and emission in the visible region with large Stokes shifts. Probes featuring electron-donating groups exhibit rotor effects that are sensitive to polarity and viscosity by "intramolecular charge transfer" (ICT) and twisted intramolecular charge transfer (TICT) mechanisms, respectively. These properties enable their application as "turn-on" fluorescent probes to detect fibrillar aggregates of the α-synuclein (αS) protein that are a hallmark of Parkinson's disease (PD). One probe shows selective binding to αS fibrils relative to soluble proteins in cell lysates and amyloid fibrils of tau and amyloid-ß. Finally, we demonstrate the diagnostic potential of the probe in selectively detecting αS fibrils amplified from PD with dementia (PDD) patient samples.

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer.

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

Engineered RecA Constructs Reveal the Minimal SOS Activation Complex.

The SOS response is a bacterial DNA damage response pathway that has been heavily implicated in bacteria's ability to evolve resistance to antibiotics. Activation of the SOS response is dependent on the interaction between two bacterial proteins, RecA and LexA. RecA acts as a DNA damage sensor by forming lengthy oligomeric filaments (RecA*) along single-stranded DNA (ssDNA) in an ATP-dependent manner. RecA* can then bind to LexA, the repressor of SOS response genes, triggering LexA degradation and leading to induction of the SOS response. Formation of the RecA*-LexA complex therefore serves as the key "SOS activation signal." Given the challenges associated with studying a complex involving multiple macromolecular interactions, the essential constituents of RecA* that allow LexA cleavage are not well defined. Here, we leverage head-to-tail linked and end-capped RecA constructs as tools to define the minimal RecA* filament that can engage LexA. In contrast to previously postulated models, we found that as few as three linked RecA units are capable of ssDNA binding, LexA binding, and LexA cleavage. We further demonstrate that RecA oligomerization alone is insufficient for LexA cleavage, with an obligate requirement for ATP and ssDNA binding to form a competent SOS activation signal with the linked constructs. Our minimal system for RecA* highlights the limitations of prior models for the SOS activation signal and offers a novel tool that can inform efforts to slow acquired antibiotic resistance by targeting the SOS response.

One- and Two-Photon Uncaging of Carbon Monoxide (CO) with Real-Time Monitoring: On-Demand Carbazole-Based Dual CO-Releasing Platform to Test over Single and Combinatorial Approaches for the Efficient Regression of Orthotopic Murine Melanoma In Vivo.

Herein, we report three new metal-free, photochemically active single, dual, and combinatorial CORMs (photoCORMs) based on a carbazole-fused 1,3-dioxol-2-one moiety which released one equivalent of CO, two equivalent of CO, and a combination of one equivalent of each CO and anticancer drug upon one- and two-photon excitation, respectively. The photoCORMs exhibited good cellular uptake and real-time monitoring ability of CO uncaging by a color change approach in cancerous B16F10 cells. Interestingly, the cytotoxicity assay on B16F10 cells indicated that the dual photoCORM has increased anticancer activity over the single and combinatorial photoCORMs upon irradiation. Our results also showed that CO could accelerate the effectiveness of the well-known anticancer drug (chlorambucil). Finally, the in vivo evaluation of the dual photoCORM on an established murine melanoma tumor (C57BL/6J mouse model) manifested a significant regression of tumor volume and led to significant improvement (>50%) in the overall survivability.

An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET.

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.

Protein labeling for FRET with methoxycoumarin and acridonylalanine.

Site-specific protein labeling can be used to monitor protein motions and interactions in real time using Förster resonance energy transfer (FRET). While there are many fluorophores available for protein labeling, few FRET pairs are suitable for monitoring intramolecular protein motions without being disruptive to protein folding and function. Here, we describe the synthesis and use of a minimally perturbing FRET pair comprised of methoxycoumarin maleimide (Mcm-Mal) and acridonylalanine (Acd). Acd can be incorporated into a protein through unnatural amino acid mutagenesis. Mcm-Mal is fluorogenic when reacted with cysteine and can label cysteine/Acd double mutant proteins. This labeling strategy provides an easy to install FRET pair with a working range or 15-40Å, making it ideal for monitoring most intramolecular motions. Additionally, Mcm/Acd FRET can be combined with tryptophan fluorescence for monitoring multiple protein motions via three color FRET.

An improved tumor-specific therapeutic strategy by the spatio-temporally controlled in situ formation of a Cu(ii) complex, leading to prompt cell apoptosis via photoactivation of a prodrug.

The anti-tumor activity of Cu complexes is well established in cancer research. We developed a biotin-tagged Cu-chelating prodrug that is activated by one-photon and two-photon irradiation for the target-specific and spatio-temporally controlled in situ generation of a Cu complex. In this way, we transform copper from a "cancer-promoting" agent to an anticancer agent.

Organophotoredox-Mediated Amide Synthesis by Coupling Alcohol and Amine through Aerobic Oxidation of Alcohol.

The combination of an organic photocatalyst [4CzIPN (1,2,3,5-tetrakis(carbazol-9-yl)-4,6 dicyanobenzene) or 5MeOCzBN (2,3,4,5,6-pentakis(3,6-dimethoxy-9 H-carbazol-9-yl)benzonitrile)], quinuclidine, and tetra-n-butylammonium phosphate (hydrogen-bonding catalyst) was employed for amide bond formations. The hydrogen-bonded OH group activated the adjacent C-H bond of alcohols towards hydrogen atom transfer (HAT) by a radical species. The quinuclidinium radical cation, generated through single-electron oxidation of quinuclidine by the photocatalyst, employed to abstract a hydrogen atom from the α-C-H bond of alcohols selectively due to a polarity effect-produced α-hydroxyalkyl radical, which subsequently converted to the corresponding aldehyde under aerobic conditions. Then the coupling of the aldehyde and an amine formed a hemiaminal intermediate that upon photocatalytic oxidation produced the amide.

Wavelength-Orthogonal Photocleavable Monochromophoric Linker for Sequential Release of Two Different Substrates.

A wavelength-orthogonal photocleavable monochromophoric linker was developed that is based on a 3-acetyl-9-ethyl-6-methylcarbazole (AEMC) moiety substituted at both the phenacyl and benzylic positions with different carboxylic acids. The different carboxylic acids were released sequentially upon irradiation with light of λ ≥ 365 nm and λ ≥ 290 nm, respectively.

Real-time monitoring of a photoactivated hydrogen persulfide donor for biological entities.

Hydrogen persulfide (H2S2) plays an important role in sulfur-based redox signaling mechanisms. Herein, we developed a visible light activated ESIPT based H2S2 donor using a p-hydroxyphenacyl phototrigger. The unique feature of the designed H2S2 donor system is the ability to monitor the H2S2 release in real time through a non-invasive fluorescence color change approach, with the color changing from green to blue. Next, we demonstrated the detection and quantification of H2S2 using a fluorescein based "turn-on" fluorescent probe. Furthermore, in vitro studies of the designed H2S2 donor demonstrated the real-time monitored H2S2 release and cytoprotective ability in the highly oxidizing cellular environment of MDA-MB-468 cells.

One- and Two-Photon-Activated Cysteine Persulfide Donors for Biological Targeting.

Persulfides have been considered as potential signaling compounds similar to the H2S in "S-persulfidation", a sulfur-mediated redox cycle. The research of this sulfur-mediated species is hindered because of the lack of efficient persulfide donors. In this current study, we have developed one- and two-photon-activated persulfide donors based on an o-nitrobenzyl (ONB) phototrigger, which releases the biologically active persulfide (N-acetyl l-cysteine persulfide, NAC-SSH) in a spatiotemporal manner. Next, we have demonstrated the detection of persulfide release both qualitatively and quantitatively using the well-known "turn on" fluorescence probe, that is, monobromobimane, and the trapping agent, that is, 2,4-dinitrofluorobenzene, respectively. Furthermore, we examined the cytotoxicity of synthesized persulfide donors on HeLa cells and the cytoprotective ability in the highly oxidizing cellular environment.

One- and two-photon responsive sulfur dioxide (SO2) donors: a combinatorial drug delivery for improved antibiotic therapy.

One- and two-photon activated sulfur dioxide donors based on a 4,5-dimethoxy-2-nitrobenzyl phototrigger have been developed. The designed donors have the ability to release not only SO2 but also a hydroxy-compound in a simultaneous manner. Furthermore, we demonstrated their application in combinatorial therapy by the dual release of SO2 and an active drug, i.e. ferulic acid ethyl ester (FAEE) with self-monitoring ability. Next, we investigated the in vitro cellular uptake and the capability of SO2 generation from the donors using a well-known coumarin-hemicyanine fluorescent probe. Finally, we evaluated the antibacterial activity of the designed donors (5a, 5b and 6) by broth dilution and agar well diffusion methods on E. cloacae cells (MTCC 509). The results show that the donor 5a exhibits enhanced antibacterial efficacy compared to 5b and 6, due to the synergetic effect of dually released SO2 and FAEE.

Metallaphotoredox-Mediated Csp2-H Hydroxylation of Arenes under Aerobic Conditions.

The direct hydroxylation of 2-arylpyridines and 2-arylbenzothiazoles via the merger of organic photoredox and metal catalysis is reported where 4CzIPN is used as the visible-light photocatalyst and Pd(OAc)2 as the metal catalyst. This method has been employed to synthesize organic molecules exhibiting excited-state intramolecular proton transfer properties for generating tunable luminescence responses.

One- and Two-Photon Uncaging: Carbazole Fused o-Hydroxycinnamate Platform for Dual Release of Alcohols (Same or Different) with Real-Time Monitoring.

A one- and two-photon activated photoremovable protecting group (PRPG) was designed based on a carbazole fused o-hydroxycinnamate platform for the dual (same or different) release of alcohols. The mechanism for the dual release proceeds through a stepwise pathway and also monitors the first and second photorelease in real time by an increase in fluorescence intensity and color change, respectively. Further, its application in staining live neurons and ex vivo imaging with two-photon excitation is shown.

Light triggered uncaging of hydrogen sulfide (H2S) with real-time monitoring.

An ESIPT based light activated hydrogen sulfide (H2S) donor using a p-hydroxyphenacyl phototrigger has been developed. The unique feature of our H2S donor system is that it provides real-time monitoring of H2S release by a non-invasive fluorescence colour change approach.

Cascade photocaging of diazeniumdiolate: a novel strategy for one and two photon triggered uncaging with real time reporting.

We report a new strategy, viz. cascade photocaging, for protecting diethylamine diazeniumdiolate (O2-position), a light sensitive molecule. Upon photolysis, the cascade photocage at first releases the light activatable linker (latent fluorophore) O2-caged diazeniumdiolate, which undergoes spontaneous 1,8-elimination, triggering the release of the diazeniumdiolate anion and the fluorophore.

Redox-responsive xanthene-coumarin-chlorambucil-based FRET-guided theranostics for "activatable" combination therapy with real-time monitoring.

A FRET donor-acceptor xanthene-coumarin conjugate has been designed for redox-regulated synergic treatment of photodynamic therapy and chemotherapy with real-time monitoring. The "locked" FRET pair was selectively "unlocked" by biological reducing thiols via rupture of a sacrificial disulfide linker. A distinct change in fluorescence color and selective cancer cell toxicity were observed in vitro.

Bimane: A Visible Light Induced Fluorescent Photoremovable Protecting Group for the Single and Dual Release of Carboxylic and Amino Acids.

A series of ester conjugates of carboxylic and amino acids were synthesized based on bimane fluorescent photoremovable protecting group (FPRPG). The photorelease of single and dual (same as well as different) carboxylic and amino acids is demonstrated from a single bimane molecule on irradiation with visible light (λ ≥ 410 nm). The detailed mechanistic study of photorelease revealed that the release of two caged acids is simultaneous but in a stepwise pathway.