Resonance Energy Transfer
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2021 ◽  
Tomozumi Imamichi ◽  
John G. Bernbaum ◽  
Sylvain Laverdure ◽  
Jun Yang ◽  
Qian Chen ◽  

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy naïve patients reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus drugs naïve patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I forms homodimer with a similar efficiency with GagPol (WT). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase or Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are not only regulated by integrase but also RNase H. Importance A nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Co-existing other SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

2021 ◽  
Vol 22 (18) ◽  
pp. 9945
Luisa Galla ◽  
Nicola Vajente ◽  
Diana Pendin ◽  
Paola Pizzo ◽  
Tullio Pozzan ◽  

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1825
Yue Zhang ◽  
Huijie Chen ◽  
Mengmeng Zou ◽  
Rick Oerlemans ◽  
Changhao Shao ◽  

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

2021 ◽  
Vol 8 ◽  
Taryn M. Kay ◽  
Cody P. Aplin ◽  
Rowan Simonet ◽  
Julie Beenken ◽  
Robert C. Miller ◽  

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.

2021 ◽  
Yongjun Zheng ◽  
Hong Yang ◽  
Lufang Zhao ◽  
Yuhan Bai ◽  
Xinghua Chen ◽  

By virtue of near-zero optical background and photobleaching, electrochemiluminescence (ECL), an optical phenomenon excited by electrochemical reactions, has drawn extensive attention in both fundamental studies and wide applications especially of ultrasensitive bioassay. Developing diverse ECL emitters is crucial to unlock their multiformity and performances, but remains a formidable challenge, due to the rigorous requirements for ECL. Herein, we report a general intramolecular ECL resonance energy transfer (iECL-RET) strategy to light up ECL-inactive dyes in aqueous solutions using an existing high-performance ECL initiators. As a proof-of-concept, a series of luminol donor-dye acceptor based ECL emitters with near unity RET efficiency and coarse/fine tunable emission wavelengths were demonstrated. Different to previous exploitation of new molecule single-handedly to address all the prerequisites of ECL, each unit in the proposed ECL ensemble performed maximally its own functions. The iECL-RET strategy would greatly expand the family members of ECL emitters for more demanding future applications.

2021 ◽  
Katherina Hemmen ◽  
Susobhan Choudhury ◽  
Mike Friedrich ◽  
Johannes Balkenhol ◽  
Felix Knote ◽  

We present a protocol and workflow to perform live cell dual-color fluorescence crosscorrelation spectroscopy (FCCS) combined with Förster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represents the dynamical "fingerprint" of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known "nanoruler" to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations, and mobility constants become accessible in cellular settings. Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerable sample itself. We will show how to perform the experiments including the calibration steps. We use dual-color labeled β2-adrenergic receptor (β2AR) labeled (eGFP and SNAPtag-TAMRA). We will guide you step-by-step through the data analysis procedure using open-source software and provide templates that are easy to customize. Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis near-physiological conditions.

2021 ◽  
Vol 11 (1) ◽  
Irina Starostina ◽  
Yoon-Kwan Jang ◽  
Heon-Su Kim ◽  
Jung-Soo Suh ◽  
Sang-Hyun Ahn ◽  

AbstractTransient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca2+ channel, plays a crucial role in intracellular Ca2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca2+ influx at plasma membrane microdomains. Using two different types of Ca2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels.

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