Intramolecular Fluorescence Resonance Energy Transfer Research Articles

A coumarin-naphthalimide-based ratiometric fluorescent probe for nitroxyl (HNO) based on an ICT-FRET mechanism

Nitroxyl (HNO) is an important reactive nitrogen that is associated with various states in physiology and pathology and plays a unique function in living systems. So, it is important to exploit fluorescent probes with high sensitivity and selectivity for sensing HNO. In this paper, a novel ratiometric fluorescent probe for HNO was developed utilizing intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. The probe selected coumarin as energy donor, naphthalimide as energy receptor and 2-(diphenylphosphino)benzoate as the sensing site for detecting HNO. When HNO was not present, the 2-(diphenylphosphino)benzoate unit of the probe restricted electron transfer and the ICT process could not occur, leading to the inhibition of FRET process as well. Thus, in the absence of HNO the probe displayed the intrinsic blue fluorescence of coumarin. When HNO was added, the HNO reacted with the 2-(diphenylphosphino)benzoate unit of the probe to yield a hydroxyl group which resulting in the opening of ICT process and the occurring of FRET process. Thus, after providing HNO the probe displayed yellow fluorescence. In addition, the probe showed good linearity in the ratio of fluorescence intensity at 545 nm and 472 nm (I545 nm/I472 nm) with a concentration of HNO (0.1–20 μM). The probe processed a detection limit of 0.014 μM and a response time of 4 min. The probe also specifically identified HNO over a wide pH scope (pH = 4.00–10.00), including physiological conditions. Cellular experiments had shown that this fluorescent probe was virtually non-cytotoxic and could be applied for ratiometric sensing of HNO in A549 cells.

A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

An ICT-FRET-based ratiometric fluorescent probe for hydrogen polysulfide based on a coumarin-naphthalimide derivative

Hydrogen polysulfide (H2Sn, n > 1), as one of the important members of reactive sulfur species (RSS), plays a vital part in the processes of both their physiology and pathology. In this work, a ratiometric fluorescent probe for H2Sn had been designed and prepared based on the combination mechanism of intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET). The probe chose a coumarin derivative as the energy donor, a naphthalimide derivative as the energy acceptor and 2-fluoro-5-nitrobenzoate as the H2Sn recognition group. When H2Sn was not present in the system, the ICT process of the naphthalimide acceptor was inhibited and the FRET process from the coumarin donor to the naphthalimide acceptor was turned off. When H2Sn was added, both ICT and FRET occurred due to the nucleophilic substitution-cyclization reactions between the probe and hydrogen polysulfide. In addition, the ratio value of the emission intensities at 550 nm and 473 nm (I550 nm/I473 nm) of this probe had a good linear relationship with H2Sn concentration in the range of 6.0 × 10-7-5.0 × 10-5 mol·L-1, and a detection limit of 1.8 × 10-7 mol·L-1 was obtained. The developed probe had high selectivity and sensitivity, as well as good biocompatibility. Additionally, the probe had been used to successfully image both indigenous and exogenous hydrogen polysulfide in A549 cells using confocal microscope.

Intramolecular fluorescence resonance energy transfer strategy for accurate detection of AFP-L3% and improved diagnosis of hepatocellular carcinoma

Early and accurate diagnosis of hepatocellular carcinoma (HCC) is of significant importance for improving the survival rate and quality of life for HCC patients. The combined detection of alpha-fetoprotein (AFP) and alpha-fetoprotein-L3 (AFP-L3), namely AFP-L3%, can greatly improve the accuracy of HCC diagnosis compared with AFP detection. Herein, we developed a novel intramolecular fluorescence resonance energy transfer (FRET) strategy for sequential detection of AFP and AFP-specific core fucose to improve the diagnosis accuracy of HCC. Firstly, fluorescence-labeled AFP aptamer (AFP Apt-FAM) was used to specifically recognize all AFP isoforms, and total AFP was quantitatively determined using fluorescence intensity of FAM. Then, 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) labeled lectins (PhoSL-Dabcyl) were used to specifically recognize the core fucose expressed on AFP-L3 that does not bind to other AFP isoforms. The combination of FAM and Dabcyl on the same AFP molecule could generate FRET effect, thereby quenching the fluorescence signal of FAM and quantitatively determining AFP-L3. After that, AFP-L3% was calculated according to the ratio of AFP-L3 to AFP. With this strategy, the concentration of total AFP, AFP-L3 isoform as well as the AFP-L3% were sensitively detected. Detection limits of 0.66 and 0.186 ng/mL were obtained for AFP and AFP-L3 in human serum, respectively. Clinical human serum test results showed that AFP- L3 % test was more accurate than AFP assay to distinguish healthy people, HCC patients and benign liver disease patients. Therefore, the proposed strategy is simple, sensitive and selective, which can improve the accuracy of early diagnosis of HCC, and has good clinical application potential.

Modulating Energy Transfer and Light-Emitting Colors by Chiral Dye Exchange and Achiral-to-Chiral Dye Conversion.

New chiral and achiral dyes were synthesized by using clean, highly efficient Diels-Alder 'click' reactions. We prepared pure endo- and exo- stereoisomers and blue-light-emitting single crystal of an exo-isomer. 1 H NMR spectroscopy and single-crystal X-ray diffraction reveal the precise spatial configurations of endo- and exo-dye stereoisomers. Single-crystal X-ray diffraction results reveal the dynamic nature of Diels-Alder covalent bonds. By reversible formation and cleavage of dynamic covalent bonds, an achiral dye was converted to a chiral dye, where achiral dansyl dye moiety was replaced by chiral moiety. Before achiral-to-chiral dye conversion, the dye system displayed dansyl acceptor green light-emitting color due to intramolecular fluorescence resonance energy transfer, where the blue emission of pyrene donor fluorophore was not observed. After achiral-to-chiral dye conversion, light-emitting colors of dye system change from green to blue, where the pyrene-to-dansyl fluorophore energy transfer was suppressed, and pyrene fluorophore blue emission was restored. Chiral transfer occurs in a chiral dye solid from non-chromophore alky chirality to pyrene chromophore. Thus, the chiral dye solid displayed chiral optical behaviors, i. e., circular dichroism and circularly polarized luminescence.

Diverse Fluorescence Resonance Energy Transfer Processes Originating from the Conformational Heterogeneity of the Calcium Indicator Yellow Cameleon YC3.60.

By combining time-resolved fluorescence spectroscopy and molecular biology, we have clearly elucidated the fluorescence mechanism of yellow cameleon YC3.60. YC3.60 is one of the most widely used fundamental calcium ion indicator proteins and is a tandem protein consisting of cyan fluorescent protein, calmodulin, and yellow fluorescent protein. Our results show that the conformational heterogeneity of YC3.60 leads to multiple FRET (fluorescence resonance energy transfer) processes occurring in a variety of structures. In the presence of calcium ions, FRET occurs in 75% of all YC3.60 molecules, and the intramolecular FRET faster than 20 ps is dominant among the three different FRET processes. Even in the absence of calcium ions, FRET occurs in 28% of YC3.60, where intra- and intermolecular FRET with a time constant of 160 ps is dominant among the four different FRET processes. YC3.60 with the immature chromophore, which is 25% of the total, would result in lower contrast on imaging. Conformational heterogeneity arises from the specific association of the two fluorescent proteins. It was clearly shown that the association of fluorescent proteins has a significant impact on the fluorescence mechanism. These observations lead to the promotion of research that elucidates the relationship between the higher-order structure and function of proteins.

Poison peptides interactions with the sarcoplasmic reticulum Ca-ATPase (SERCA).

Maintaining cellular integrity requires maintenance of protein quality, so when damaged/misfolded proteins fail to be repaired, they are degraded via proteolysis. Proteolysis increases in heart disease, overwhelming the proteosome, resulting in an accumulation of protein fragments in the cell. We propose that undegraded transmembrane protein fragments found in heart failure may persist in the membrane and interact with SERCA, competing with and displacing the physiological regulatory micropeptide phospholamban. These interactions with “poison peptides” may contribute to calcium mishandling in heart failure. To test this hypothesis, we performed mass spectrometry of membrane fractions isolated from failing human myocardium. We detected approximately 1000 protein fragments of interest in the failing heart, then narrowed our selection to high-abundance transmembrane protein fragments ranging from 20-40 amino acids. We used fluorescence resonance energy transfer (FRET) to test whether seven candidate poison proteins could interact with SERCA. Some peptides did not interact with SERCA at all, some bound weakly, and some bound with a high affinity. The highest affinity candidates bound SERCA as avidly as some native regulatory micropeptides. The candidate peptides tested here include transmembrane fragments of phospholamban (PLB), phospholemman (PLM), calcium voltage-gated channel subunit alpha1 E (CACNA1E) and sarcolemma associated protein (SLMAP). We also tested whether the putative poison peptides altered the structure of SERCA, using time-correlated single photon counting to quantify intramolecular FRET in a “2-color SERCA” construct labeled with mCyRFP-1 and mMaroon1. Overall, the results suggest that transmembrane peptide fragments generated in heart failure can interact with SERCA and may disrupt calcium handling in the failing heart.

3-Hydroxyflavone - bovine serum albumin complex used as fluorescent probe for the detection of pepsin

In this study, a method for the determination of pepsin using a 3-hydroxyflavone (3-HF)-bovine serum albumin (BSA) complex as a fluorescent probe was established. Under acidic conditions, 3-HF can react with BSA to emit strong green fluorescence, which involves an excited state intramolecular proton transfer and fluorescence resonance energy transfer. After pepsin is added, the fluorescence of the 3-HF-BSA system is quenched due to the hydrolysis of BSA. The degree of 3-HF-BSA fluorescence quenching has a good linear relationship with the concentration of pepsin in the range of 10–80 μg/mL. The correlation coefficient was R2 = 0.9932 and the detection limit was 2.41 μg/mL. This method has high selectivity and good stability and has been used to determine pepsin in real samples with satisfactory results.

Chiral smart bionic skin film with changeable structural colors and tunable luminescence by polymer-assisted supramolecular assembly of the photonic crystals

The biological skin system of advanced organisms can obtain various environmental stimuli to change skin colors by adjusting the micro-nano structure of the skin surface or by electroluminescence and photoluminescence. The assembly of biominetic skin materials with both changeable structural colors and tunable luminescence is a significant challenge in the fields of advanced materials and light science. This work describes a novel chiral smart bionic skin film with changeable structural colors, tunable luminescence by the polymer-assisted supramolecular assembly of the photonic crystals. A smart nanodot based on the molecule with four-armed mimosa-like structure was assembled on the cellulose nanocrystals (CNC) photonic crystal. The chiral bionic skin film obtained had multi-channel fluorescence-emission, sensitive structural color stimulus responses to the light and humidity and the tunable chiral helical nematic domains. It demonstrates a series of the supramolecular interactions that trigger multiple intelligent signals, including the adjustable chiral pitch, photo-induced electron transfer (PET), intramolecular fluorescence resonance energy transfer (FRET), π-π stacking and the changes of the hydrogen bonds. The photonic crystal skin materials with sensitive multi-signals intelligent responses as smart bionic skin have potential applications for the tissue engineering, 3D printing materials, bionic artificial skin materials, and optical devices.

A pyrene-derived ratiometric fluorescent probe for pH monitoring in cells and zebrafish based on monomer-excimer emission

Acidity in biological microenvironments plays a crucial role in physiology and pathology, so that pH monitoring can provide insights into the mechanisms underlying pH-related processes. Many pH ratiometric fluorescent probes have been developed based on intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) principles. In contrast, those based on intermolecular excimer are scarce. The present work rationally designed and readily synthesized a pyrene-derived ratiometric fluorescent probe (Pyrene-PA) for pH detection using the conversion of monomer-excimer emission. In an acidic aqueous medium, Pyrene-PA exists in a cationic monomer with an emission centered at 463 nm. As pH increases, Pyrene-PA converts to neutral molecules and exhibits reduced dispersibility, generating an intense excimer emission around 630 nm. Based on the above mechanism, Pyrene-PA displays a sensitive, selective, photostable, and reversible response to pH changes without interference from coexisting species such as anions, cations, thiols, and reactive oxygen species. The ratio of fluorescence intensity at 630 nm and 463 nm increases linearly with pH values from 4.0 to 7.0, and the pKa value is 5.6, suitable for pH quantification in acidic to neutral environments. Moreover, Pyrene-PA with low cytotoxicity and good cell penetrability enables monitoring pH changes in cells and zebrafish.

Apoptosis Detection in Retinal Ganglion Cells Using Quantitative Changes in Multichannel Fluorescence Colocalization.

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma.

Self-Assembled Nanodot Actuator with Changeable Fluorescence by π-π Stacking Force Based on a Four-Armed Foldable Phthalocyanine Molecule and Its Supersensitive Molecular Recognition.

Designing intelligent molecules and smart nanomaterials as molecular machines is becoming increasingly important in the nanoscience fields. Herein, we report a nanodot actuator with changeable fluorescence by π-π stacking force based on a four-armed foldable phthalocyanine molecule. The assembled nanodot possessed a three-dimensional molecular space structure and multiple supramolecular interactions. The arms of the nanodot could fold and open intelligently in response to environmental molecular stimuli such as natural plant mimosa, which could lead to multiple variable fluorescence emissions. The nanodot was highly sensitive to the biomolecule thyroxine at the molecular level. The accurate molecular recognition and the changeable fluorescence conversion of the nanodot were attributed to multiple supramolecular interactions, including photoinduced electron transfer (PET), intramolecular fluorescence resonance energy transfer (FRET), and π-π stacking of the nanodots, resulting in an intelligent "nanodot machine with folding arms". The self-assembled nanodot actuators with changeable fluorescence have potential applications in advanced intelligent material fields.

Enzyme-Activated Multifunctional Prodrug Combining Site-Specific Chemotherapy with Light-Triggered Photodynamic Therapy.

Combination treatments are more effective than conventional monotherapy in combating cancer. Herein, a multifunctional prodrug BDP-L-CPT was rationally engineered and prepared by the conjugation of a boron dipyrromethene (BDP)-based photosensitizer (PS) to the active site of the chemotherapeutic drug camptothecin (CPT) via a phenyl benzoate group. After modification, the cytotoxicity of CPT was locked. Moreover, the fluorescence emission at 430 nm from the CPT component in the prodrug was substantially inhibited through the intramolecular fluorescence resonance energy transfer process. The phenyl benzoate linker in BDP-L-CPT could be selectively cleaved by exogenous carboxylesterase in phosphate-buffered saline solution and endogenous carboxylesterase overexpressed in cancer cells, which was followed by self-immolation to release free CPT. The drug release process could be monitored by the turn-on of CPT fluorescence in solution and cells. Owing to the combination of site-specific chemotherapy with light-driven photodynamic therapy, the IC50 values of the prodrug BDP-L-CPT against HepG2 human hepatocellular carcinoma and HeLa human cervical carcinoma cells were lower than those of the controls, BDP-COOH and CPT. The combined antitumor effects of the prodrug BDP-L-CPT were also observed in the mice bearing H22 tumors. Furthermore, BDP-L-CPT had a more prolonged blood circulation time in mice than CPT, which is beneficial to persistent therapy. This study may provide a promising strategy for a selective combination cancer treatment by conjugating a prodrug to a PS.

General Strategy to Achieve Color-Tunable Ratiometric Two-Photon Integrated Single Semiconducting Polymer Dot for Imaging Hypochlorous Acid.

It is highly desired and challenging to construct integrated (all-in-one) single semiconducting-polymer-derived dot (Pdot) without any postmodification but with desired performances for bioapplications. In this work, eight hypochlorous acid (HClO)-sensitive integrated polymers and corresponding polymer-derived Pdots are designed through molecular engineering to comparatively study their analytical performances for detecting and imaging HClO. The optimized polymers-derived Pdots are obtained through regulating donor-acceptor structure, the content of HClO-sensitive units, and the position of HClO-sensitive units in the polymer backbone. The designed Pdots display distinguished characteristics including multicolours with blue, yellow, and red three primary fluorescence colors, determination mode from single-channel to dual-channel (ratiometric) quantification, ultrafast response, low detection limit, and high selectivity for ClO- sensing based on specific oxidation of ClO--sensitive unit 10-methylphenothiazine (PT) accompanied by altering the intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) processes in Pdots. The prepared integrated Pdots are also applied for two-photon ClO- imaging in HeLa cells and one- and two-photon ClO- imaging produced in acute inflammation in mice with satisfactory results. We believe that the present study not only provides excellent integrated fluorescent nanoprobes for ClO- monitoring in living systems but also extends a general strategy for designing integrated semiconducting polymers and Pdots with desired performances for biological applications.

Coupling Aptamer‐based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein‐Specific Glycosylation

AbstractExosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein‐specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD‐L1 (exoPD‐L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD‐L1 aptamers bound on exoPD‐L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD‐L1 glycosylation was confirmed to be required in interaction with PD‐1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non‐destructive method to study the presence and function of exosomal protein‐specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.

Coupling Aptamer-based Protein Tagging with Metabolic Glycan Labeling for In Situ Visualization and Biological Function Study of Exosomal Protein-Specific Glycosylation.

Exosomal glycoproteins play important roles in many physiological and pathological functions. Herein, we developed a dual labeling strategy based on a protein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes. The glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling. This method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD-L1 glycosylation was confirmed to be required in interaction with PD-1 and participated in inhibiting of CD8+ T cell proliferation. This is an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides a powerful tool for exosomal glycoproteomics research.

Methods, principles and applications of optical detection of metal ios

• The optical detection methods of 12 common metal ions are reviewed. • The main principles and experimental phenomena of optical detection are summarized. • The development and advantages of optical detection methods are summarized. • The applications of optical detection method are summarized. • The future development and main challenges of optical detection are discussed. Heavy metal elements were widely used in alloy manufacturing, medical treatment, food additive, military industry and other fields, which were harmful to human body. Optical detection was a non-contact nondestructive detection method, which had the advantages of high selectivity, high sensitivity and low cost, and had important applications and prospects in environmental monitoring and biological science. In this paper, the optical detection methods, detection principles and detection methods of 12 kinds of metal ions (Ag + , Al 3+ , Cd 2+ , Co 2+ , Cr 3+ , Cu 2+ , Fe 2+ , Fe 3+ ,Hg 2+ ,Pb 2+ , Pd 2+ ,Zn 2+ ) were reviewed. Fluorescence, colorimetry and spectral change were the main detection methods. Fluorescence detection methods mainly included fluorescence quenching, fluorescence enhancement and fluorescence spectrum changes; The colorimetric method included the change of color and the change of ultraviolet absorption spectrum; The spectral changes mainly included circular dichroism, phosphorescence and surface enhanced Raman scattering, etc. Photoinduced electron transfer, intramolecular charge transfer and fluorescence resonance energy transfer were the primary principles of optical detection. The application prospect and main challenges of optical detection were discussed.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein.

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, etc. The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.

Time-resolved detection of SDS-induced conformational changes in α-synuclein by a micro-stopped-flow system.

An intrinsically disordered protein, α-synuclein (αSyn), binds to negatively charged phospholipid membranes and adopts an α-helical structure. This conformational change is also induced by interaction with sodium dodecyl sulfate (SDS), which is an anionic surfactant used in previous studies to mimic membrane binding. However, while the structure of the αSyn and SDS complex has been studied widely by various static measurements, the process of structural change from the denatured state to the folded state remains unclear. In this study, the interaction dynamics between αSyn and SDS micelles was investigated using time-resolved measurements with a micro-stopped-flow system, which has been recently developed. In particular, the time-resolved diffusion based on the transient grating technique in combination with a micro-stopped-flow system revealed the gradual change in diffusion triggered by the presence of SDS micelles. This change is induced not only by binding to SDS micelles, but also by an intramolecular conformational change. It was interesting to find that the diffusion coefficient decreased in an intermediate state and then increased to the final state in the binding reaction. We also carried out stopped-flow-kinetic measurements of circular dichroism and intramolecular fluorescence resonance energy transfer, and the D change was assigned to the formation of a compact structure derived from the helix bending on the micelle.