Single-molecule fluorescence resonance energy transfer in molecular biology

Sasmal, Dibyendu Kumar; Pulido, Laura E.; Kasal, Shan; Huang, Jun

doi:10.1039/c6nr06794h

Cited by 95 publications

(93 citation statements)

References 136 publications

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“…Since its first demonstration in 1996, smFRET has been used to provide mechanistic answers in diverse areas of biological research. These studies unraveled molecular mechanisms of helicases and topoisomerases (40), DNA replication, DNA repair (41), transcription (42–44), translation (42,45,46), enzymatic function (47–49), molecular motors (50), membrane proteins (51), protein folding (52, 53), nucleic acids (54, 55), RNA folding (54, 56, 57), and ribozyme catalysis (58, 59). Because a short review cannot do justice to the large number and diversity of smFRET studies, we will discuss a few representative examples.…”

Section: A Brief Historical Overview Of Single-molecule Fret In Biochmentioning

confidence: 99%

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Lerner

Cordes

Ingargiola

et al. 2018

Science

489

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Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

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Section: A Brief Historical Overview Of Single-molecule Fret In Biochmentioning

confidence: 99%

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Lerner

Cordes

Ingargiola

et al. 2018

Science

489

449

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“…FRET is a nonradiative process and originated by dipole-dipole interaction between the electronic states of donor and acceptor 8,40 The energy transfer occurs only when the oscillations of an optically-induced electronic coherence of the donor are resonant with the electronic energy gap of the acceptor. Efficiency of energy transfer ( E FRET ) is sensitive to the inter-distance between the donor and acceptor, which is typically in the range of 10-100 Å.…”

Section: Methodsmentioning

confidence: 99%

“…Efficiency of energy transfer ( E FRET ) is sensitive to the inter-distance between the donor and acceptor, which is typically in the range of 10-100 Å. The energy transfer efficiency ( E FRET ) is given by 8,40 :…”

Section: Methodsmentioning

confidence: 99%

“…However, crystal structures only provide a “snapshot” of the thermodynamically stable conformation of purified TCR and pMHC proteins. To carefully examine possible TCR conformational changes and critically probe the mechanisms of TCR ligand discrimination in situ , we used FRET 5,8 , a spectroscopic ruler with subnanometer precision, to measure the intermolecular length of a TCR-pMHC bond and the intramolecular distance of a TCR-CD3ζ complex at the immunological synapse of a live primary 5C.C7 transgenic CD4 + T cell in real time with a temporal resolution of ~10 ms.…”

Section: Figmentioning

confidence: 99%

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TCR-pMHC bond length controls TCR ligand discrimination

Sasmal

Feng

S³

et al. 2018

Preprint

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12T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-1 major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-2 pMHCs to trigger antigen-specific adaptive immune responses [1][2][3][4] . Despite intense efforts, the 3 mechanism underlying TCR ligand discrimination remains a major unanswered question in 4 immunology. Here we show that a TCR discriminates between closely related peptides by 5 forming TCR-pMHC bonds with different lengths, which precisely control the accessibility 6 of CD3z immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. 7Using in situ fluorescence resonance energy transfer (FRET) 3,5 , we measured the 8 intermolecular length of single TCR-pMHC bonds and the intramolecular distance of 9 individual TCR-CD3z complexes at the membrane of live primary T cells. We found that an 10 agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3z off the inner 11 leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong 12 phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond 13 with the TCR, resulting in partial dissociation of CD3z from the membrane and weak 14 phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR 15 binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the 16 entire process of signal reception, transduction and regulation. Thus, our data reveal the 17 fundamental mechanism by which a TCR deciphers the structural differences between 18 foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR 19 signaling for ligand discrimination. 20It remains elusive how a TCR deciphers the structural differences among peptides and 21properly propagates surface recognition signals across the cell plasma membrane to CD3 22 intracellular ITAM domains to induce distinct T-cell signaling. The TCR conformational change and accessary molecules ICAM-1 and B7-1, respectively ( Fig. 1b-e, Extended Data Fig. 2a and 3-1 4, Supplimentary Movie 1-3). For cell surface FRET1, we readily detected FRET signals for three 2 agonist pMHCs but not for a null pMHC on lipid bilayer and glass surface, and the FRET 3 efficiencies (EFRET) were positively correlated with the pMHC potencies in activating T cells 9 . The 4 average synaptic EFRET1 was 0.79, 0.54 and 0.29 for the super agonist K5, agonist MCC and weak 5 agonist 102S, respectively (Fig. 1d). However, no synaptic FRET was found for the null pMHC 6 ( Fig. 1b and d). These data validated the specificity of our cell surface TCR-pMHC FRET and 7 were consistent with a previous report 3 . In contrast, the transmembrane TCR-CD3z FRET 8 efficiencies were inversely correlated with the pMHC potencies with the highest FRET observed 9 for the null ligand ( Fig. 1c and e). In the presence of agonist pMHCs K5, MCC and 102S, the 10 transmembrane FRET was only detected at the TCR-CD3z co-localized microclusters but not 11 o...

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“…Among these biophysical methods, smFRET measures the distance between two fluorophore dyes acting as labels attached to residues in proteins or nucleotides to study protein folding, proteinprotein interaction and protein-DNA/RNA interaction (Sasmal et al, 2016). However, these distances suffer from uncertainties due to the effect of the local environment on the dye pairs (Dimura et al, 2016;Kalinin et al, 2012;Muschielok et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

DR-SIP: Protocols for Higher Order Structure Modeling with Distance Restraints- and Cyclic Symmetry-Imposed Packing

Chan

Zou

Chien

et al. 2018

Preprint

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MotivationQuaternary structure determination for proteins is difficult especially for transmembrane proteins. Even if the monomeric constituents of complexes have been experimentally resolved, computational prediction of quaternary structures is a challenging task particularly for higher order complexes. It is essential to have a reliable computational protocol to predict quaternary structures of both transmembrane and soluble proteins leveraging experimentally determined distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins. ResultsWe survey 115 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) to discover that 90% of them are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) filter and the Symmetry-Imposed Packing (SIP) filter which takes advantage of the statistically derived tilt angle cutoff and the Cn symmetry of HoTPs without prior knowledge of the number ("n") of monomers. Using only the geometrical filter, SIP, near-native poses of the 115 HoTPs can be correctly identified in the top-5 for 52% of all cases, or 49% among the HoTPs having an n >2 (~60% of the dataset), while ZDOCK alone returns 41% and 24%, respectively. Applying only SIP to three HoTPs with distance restraints, the near-native poses for two HoTPs are ranked 1 st and the other 7 th among 54,000 possible decoys. With both filters, the two remain 1 st while the other improved to 2 nd . While a soluble system with distance restraints is recovered at the 1 st -ranked pose by applying only DR. Availability and Implementationhttps://github.com/capslockwizard/drsip Supplementary informationSupplementary methods and results are available.

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Single-molecule fluorescence resonance energy transfer in molecular biology

Cited by 95 publications

References 136 publications

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

TCR-pMHC bond length controls TCR ligand discrimination

DR-SIP: Protocols for Higher Order Structure Modeling with Distance Restraints- and Cyclic Symmetry-Imposed Packing

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