Fluorescence Anisotropy Assay Research Articles

Polycations as Aptamer-Binding Modulators for Sensitive Fluorescence Anisotropy Assay of Aflatoxin B1.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer-fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Thwarting resistance: MgrA inhibition with methylophiopogonanone a unveils a new battlefront against S. aureus

Limitations in the clinical treatment of Staphylococcus aureus (S. aureus) infections have arisen due to the advent of antibiotic-resistant strains. Given the immense potential of therapeutic strategies targeting bacterial virulence, the role of MgrA as a pivotal virulence determinant in S. aureus-orchestrating resistance, adherence, and hundreds of virulence targets—becomes indispensable. In this investigation, leveraging advanced virtual screening and fluorescence anisotropy assays, we discerned methylophiopogonanone A (Mo-A), a flavonoid derivative, as a potent disruptor of the MgrA-DNA interaction nexus. Subsequent analysis revealed that Mo-A effectively inhibits the expression of virulence factors such as Hla and Pvl in S. aureus and markedly reduces its adhesion capability to fibrinogen. On a cellular landscape, Mo-A exerts a mitigating influence on the deleterious effects inflicted by S. aureus USA300 on A549 cells. Furthermore, our data indicate that Mo-A downregulates the transcription of genes associated with immune evasion, such as nucleases (nuc), Staphylococcal Chemotaxis Inhibitory Protein (chips), and Staphylococcal Complement Inhibitor (scin), thereby undermining immune escape and amplifying neutrophil chemotaxis. Upon application in an in vivo setting, Mo-A assumes a protective persona in a murine model of S. aureus USA300-induced pneumonia and demonstrates efficacy in the Galleria mellonella infection model. Of note, S. aureus displayed no swift acquisition of resistance to Mo-A, and the effect was synergistically enhanced when used in combination with vancomycin. Our findings add substantive weight to the expanding field of virulence-targeted therapeutic strategies and set the stage for more comprehensive exploration of Mo-A potential in combating antibiotic-resistant S. aureus.

A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.

The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN)and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.

The RNA-Binding Domain of hnRNP U Extends beyond the RGG/RG Motifs.

Heterogeneous nuclear ribonucleoprotein U (hnRNP U) is a ubiquitously expressed protein that regulates chromatin architecture through its interactions with numerous DNA, protein, and RNA partners. The RNA-binding domain (RBD) of hnRNP U was previously mapped to an RGG/RG motif within its disordered C-terminal region, but little is understood about its binding mode and potential for selective RNA recognition. Analysis of publicly available hnRNP U enhanced UV cross-linking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing 11 of these identified cross-link sites for biochemical characterization using a combination of fluorescence anisotropy and electrophoretic mobility shift assays. These in vitro binding experiments with a rationally designed set of RNAs and hnRNP U domains revealed that the RGG/RG motif is a small part of a more expansive RBD that encompasses most of the disordered C-terminal region. This RBD contains a second, previously experimentally uncharacterized RGG/RG motif with RNA-binding properties comparable to those of the canonical RGG/RG motif. These RGG/RG motifs serve redundant functions, with neither serving as the primary RBD. While in isolation, each RGG/RG motif has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling the binding of most of the designed RNA set with low to midnanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlight the perils of a reductionist approach to defining biochemical activities in this system and pave the way for a detailed investigation of its RNA-binding specificity.

In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.

Preclinical Characterization of AZD9574, a Blood-Brain Barrier Penetrant Inhibitor of PARP1.

We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.

Distinct membrane perturbation effects of Catestatin and its CST-364 S variant: Insights from molecular simulations and anisotropy measurements

Catestatin (CST), a versatile 21 amino acid long cationic peptide, is stored in chromaffin granules and exocytosed upon fusion with the plasma membrane. CST, produced by chromaffin cells and neutrophils, is derived from the processing of chromogranin A and induced in the skin after injury. It involves catecholamine inhibition, blood pressure control, inflammation, and innate immunity. It is thought that calcium influx is triggered by CST permeating within neutrophils. It is unknown whether CST can disturb the immediate environment enough to penetrate the cell membrane passively. We used molecular dynamics simulations to examine the behaviour of human CST in its wild-type state (CST WT) and one of its naturally occurring variants, CST-364 S, which has a high allelic frequency in the human population. Both peptides were incorporated into the model eukaryotic cell membrane known as the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) lipid bilayer. The molecular modelling and simulations results show that CST WT and CST-364 S have different propensities for membrane disruption. It was shown that CST-364 S has higher membrane permeability than CST WT. In addition, we have used fluorescence anisotropy and leakage assay to study the interaction of peptides with PC membranes. Both peptides interacted with POPC and DOPC membranes, while CST-364 S penetrated the membrane more deeply via disorganizing the membrane interface, which supports our previous findings. According to this study, there is a good possibility that the peptides will passively permeate the cell membrane, distort, and pass through it.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3Rcells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy.

The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H2O2 treatments. We found that treating cells with H2O2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H2O2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H2O2. Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H2O2-based signaling and transcription regulation roles.

Dual functions of the SARS-CoV-2 nucleocapsid domains mediate RNA binding and phase separation.

The primary purpose of the nucleocapsid (N) protein of the Sars-CoV-2 coronavirus is to interact with viral RNA and drive the formation of the viral capsid, mediated by multivalent interaction of both the N- and C-terminal domains (NTD and CTD) of N with RNA. N additionally forms liquid-liquid phase separated droplets with RNA, both in vitro and in infected cells, in a process believed to be important to viral transcription and replication. The degree to which N phase separates with RNA is dependent on the specific RNA being tested, suggesting that the secondary structure of the RNA plays a role in the propensity for phase separation. The ∼32 kb CoV-2 genomic RNA contains a heterogeneous mix of paired and unpaired bases, and while studies suggest that paired RNA is the primary driver of phase separation, little is known at the structural level about how different RNA structures interact with the nucleocapsid. Here we present investigation of binding between N and short designer oligos built to be homogenously single-stranded or double-stranded (ssRNA or dsRNA). Interestingly, we find that our 14-mer dsRNA phase separates readily with the nucleocapsid, while the ssRNA does not, indicating underlying structural differences between the two interactions. Further, we demonstrate by nuclear magnetic resonance (NMR) spectroscopy, fluorescence anisotropy and gel shift assays, that the NTD binds preferentially to ssRNA, and the CTD to dsRNA. We show that the NTD interacts nonspecifically with dsRNA, and that mutations in the NTD that disrupt RNA binding promote this nonspecific interaction. Lastly, we demonstrate that the tendency of N domains to phase separate correlates with weak, nonspecific binding interactions with RNA, suggesting that this weak, nonspecific binding is the primary driver of phase separation.

Influence of mRNA sequences downstream of a premature stop codon on nonsense suppression.

Premature stop codons (PSCs) arrest translation of full-length protein and trigger nonsense-mediated decay (NMD) of mRNA. Nonsense suppression by translational readthrough inducing drugs (TRIDs) like ataluren can restore protein function by inhibiting translation termination at PSCs catalyzed by release factor complex (RFC, eRF1.eRF3.GTP). We seek to understand what determines readthrough efficiency and apply our results to improve treatment of PSC diseases such as cystic fibrosis (CF). For this purpose, we utilize an in vitro translation system to examine the effects on RFC enzymatic activity of varying PSCs and downstream mRNA sequences in pretermination complexes, as measured with a high throughput fluorescence anisotropy assay and single-molecule TIRF microscopy. We have so far studied a total of seven sequences downstream from stop codons. Within this limited set, we find much lower EC50RFC values for sequences with stop codons UAG or UAA than for sequences having a UGA stop codon. These sequences display a ∼170-fold difference in measured EC50RFC values, a much larger range than the very limited range of RFC binding rates, 1.7-fold. We speculate that this difference is due, at least in part, to differences in the rate of reversible RFC dissociation. Additional sequences are currently under study. Our preliminary results explain, at least in part, why readthrough frequency is generally much higher for the UGA stop codon than for the UAG or UAA stop codons. They further suggest that combinations of drugs acting orthogonally to stimulate readthrough may be required to treat some PSC diseases, and that consideration of the mRNA sequence context flanking a nonsense codon might be critical for developing patient-specific therapeutic regimens.

Synthesis and evaluation of radioiodinated estrogens for diagnosis and therapy of male urogenital tumours.

The preparation of 24 estrogens, their estrogen receptor (ER) affinity and studies of radioiodinated estrogen binding to ER-positive male bladder tumor cells (HTB9) are described. The estrogens with the highest affinity were selected using fluorescence anisotropy assays. A 2,2,2-trifluoroethyl group at the 11β-position caused particularly promising affinity. (Radio)iodination was performed on the 17α-vinyl group. Binding studies on HTB9 cells revealed picomolar affinities of radioconjugates 19 and 31, indicating promising ability for targeting of urogenital tumors.

Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1.

Human phospholipid scramblase 1 (hPLSCR1) is a 37kDa multi-compartmental protein, which was initially identified as a Ca2+-dependent phospholipid translocator upon localizing to the plasma membrane. However, under certain physiological conditions, hPLSCR1 is localized to the nucleus where it interacts with the IP3R1 promoter (IP3R1P) and regulates its expression. In this study, the DNA binding properties of hPLSCR1 and ∆100-hPLSCR1 (N-terminal 100 amino acids deleted from hPLSCR1) were investigated by using a synthetic IP3R1P oligonucleotide and nonspecific scrambled-sequence oligonucleotides. Our results revealed that hPLSCR1 and ∆100-hPLSCR1 were bound to IP3R1P oligos in a 1:1 stoichiometry. In addition, ∆160-hPLSCR1 could not bind to IP3R1P oligonucleotide suggesting that the proposed DNA binding motif is the actual DNA binding motif. Specific binding of hPLSCR1 and ∆100-hPLSCR1 to IP3R1P oligos was demonstrated by fluorescence anisotropy assay. ITC analysis revealed that hPLSCR1 binds to IP3R1P oligos with Kd = 42.91 ± 0.23nM. Binding of IP3R1P oligos induces β-sheet formation in hPLSCR1 and increases the thermal stability of hPLSCR1 and ∆100-hPLSCR1. Binding of IP3R1P oligos to hPLSCR1 altered the B-form of the DNA, which was not observed with ∆100-hPLSCR1. Results from this study suggest that (i) ∆100-hPLSCR1 possesses a minimal DNA binding region and (ii) structural alterations of IP3R1P oligo by hPLSCR1 require proline-rich N-terminal region.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

Fluorescent Platforms for RNA Chemical Biology Research.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.

An Electrochemical Biosensor for Detection of P.69 Pertactin Associated with B. pertussis

Whooping cough caused by Bordetella pertussis can cause serious and prolonged health effects and is especially deadly in infants. Early treatment can significantly reduce the duration of illness and lead to better outcomes, but current diagnostic procedures need a relatively large patient sample and have an extended wait period for testing results. There is thus a pressing need for quick and accurate diagnosis. In response, we are developing an electrochemical DNA‐based (E‐DNA) biosensor for the rapid detection of B. pertussis. Our biosensor utilizes a DNA aptamer that targets P.69 pertactin, a well‐known adhesion factor in the outer membrane of B. pertussis. To generate this aptamer, we first obtained purified P.69 pertactin and biotinylated it. Biotinylation extent was measured via HABA assay and showed ~ 42 biotin molecules per P.69 pertactin molecule. This biotinylated protein was used for selection in a modified SELEX (systematic evolution of ligands by exponential enrichment) process, followed by high‐throughput sequencing. That analysis discovered six aptamer sequences with potential strong binding for P.69 pertactin. Potential aptamers were synthesized, and binding was confirmed via fluorescence anisotropy and gel shift assays. These techniques identified one aptamer sequence as having strong, low‐nanomolar binding affinity for P.69 pertactin. This aptamer was then computationally analyzed for likely secondary and tertiary structures, allowing identification of the minimum binding unit, and then was modified to generate a two‐state binding system. This modified aptamer sequence serves as the basis for an electrode‐tethered E‐DNA biosensor. This design will be verified against purified P.69 pertactin standards as well as B. pertussis isolates to demonstrate its potential as a rapid and accurate diagnostic biosensor for whooping cough infections.

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

Aptamer fluorescence anisotropy assays for detection of aflatoxin B1 and adenosine triphosphate using antibody to amplify signal change.

Fluorescence polarization/anisotropy (FP/FA) is an attractive technology for determining small molecules in homogeneous solution based on rotation changes of a fluorescent reporter. Binding induced conformation change is a specific property of aptamers. This property has been integrated into aptamer based FA assays for small molecules. In this work, we reported aptamer FA assays for aflatoxin B1 (AFB1) and adenosine triphosphate (ATP) by using antibody conjugated complementary DNA at the 3′ end and a fluorescein (FAM)-labeled aptamer at the 5′ end. The hybridization of aptamer and cDNA induced a FAM label close to the large-sized antibody, which restricts the local rotation of FAM and gives high FA signal. With the addition of target, the aptamer probe binds with the target, and the aptamer–cDNA duplex is inhibited, causing FA signal decreases. This method achieved detection of 25 pM AFB1 and 1 μM ATP, respectively. The assay is promising for application.

Intracellular artificial supramolecules based on de novo designed Y15 peptides

De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms β-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.