Selective labeling of proteins by using protein farnesyltransferase

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a F?rster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.     

Imaging of selective nuclear receptor modulator-induced conformational changes in the nuclear receptor to allow interaction with coactivator and corepressor proteins in living cells

Selective nuclear receptor modulators (SNRMs), which are used clinically for the treatment of NR-related diseases, display mixed agonistic/antagonistic activity in a tissue-selective manner depending on the cellular concentrations of coregulator proteins, that is, coactivators and corepressors. The molecular details of the SNRM function provided us with an idea for a rational method for the high-throughput screening of SNRMs in real time in intact living cells. We have developed genetically encoded fluorescent indicators based on the principle of ligand-induced coactivator and/or corepressor recruitment to NR ligand binding domain in single living cells. We demonstrated that an SNRM induces a distinct conformational change in the NR LBD, which is different from that induced by a full agonist or antagonist, but favorable for the recruitment of a coactivator or corepressor protein to the NR. The molecular details of an SNRM binding to a NR, and the subsequently induced conformational changes and recruitment of coregulator protein(s) are important features for the understanding of SNRM action in the living body. Our fluorescent indicators are capable of distinguishing among agonists, antagonists, and SNRMs, and can therefore serve as versatile molecular sensors that predict the pharmacological character of ligands, which is important for an accurate cure of a disease.     

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.     

Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5′-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.     

Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptor

We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein-protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter- or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low-noise capability of single-fluorophore detection. Using counting procedures from single-molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules.     

Identification of trinucleotide repeat ligands with a FRET melting assay

DNA hairpin structures formed within a repeated tract might be a causative factor for triplet expansion observed in several debilitating diseases. We have designed and used a fluorescence resonance energy transfer (FRET) melting assay to screen for ligands that bind specifically to the CNG triplet repeats. Using this assay, we screened a panel of 33 chemicals that were previously designed to bind DNA or RNA secondary structures. Remarkably, we found that macrocyclic compounds, such as acridine dimers and trimers, exhibit interesting affinities and specificities for this motif.     

HMGA1a protein unfolds or refolds synthetic DNA-chromophore hybrid polymers: a chaperone-like behavior

High group mobility protein, HMGA1a, was found to play a chaperone-like role in the folding or unfolding of hybrid polymers that contained well-defined synthetic chromophores and DNA sequences. The synthetic and biological hybrid polymers folded into hydrophobic chromophoric nanostructures in water, but existed as partially unfolded configurations in pH or salt buffers. The presence of HMGA1a induced unfolding of the hybrid DNA-chromophore polymer in pure water, whereas the protein promoted refolding of the same polymer in various pH or salt buffers. The origin of the chaperone-like properties probably comes from the ability of HMGA1a to reversibly bind both synthetic chromophores and single stranded DNA. The unfolding mechanisms and the binding stoichiometry of protein-hybrid polymers depended on the sequence of the synthetic polymers.     

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.     

An adaptable luminescence resonance energy transfer assay for measuring and screening protein-protein interactions and their inhibition

Protein-protein interactions (PPIs) are central to biological processes and represent an important class of therapeutic targets. Here we show that the interaction between FK506-binding protein 12 fused to green fluorescent protein (GFP-FKBP) and the rapamycin-binding domain of mTor fused to Escherichia coli dihydrofolate reductase (FRB-eDHFR) can be sensitively detected (signal-to-background ratio (S/B)>100) and accurately quantified within an impure cell lysate matrix using a luminescence resonance energy transfer (LRET) assay. Ascomycin-mediated inhibition of GFP-FKBP-rapamycin-FRB-eDHFR complex formation was also detected at high S/B ratio (>80) and Z'-factor (0.89). The method leverages the selective, stable binding of trimethoprim (TMP)-terbium complex conjugates to eDHFR, and time-resolved, background-free detection of the long-lifetime (～ms) terbium-to-GFP LRET signal that indicates target binding. TMP-eDHFR labeling can be adapted to develop high-throughput screening assays and complementary, quantitative counter-screens for a wide variety of PPI targets with a broad range of affinities that may not be amenable to purification.