Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Majumdar, Zigurts K.; Hickerson, Robyn P.; Noller, Harry F.; Clegg, Robert M.

doi:10.1016/j.jmb.2005.06.027

Cited by 79 publications

(96 citation statements)

References 70 publications

(130 reference statements)

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“…Because of this issue, controls should be performed to ensure that apparent changes in FRET are not caused by changes in the fluorophore itself (26). Previous methods to address this issue include exchanging the position of the donor and acceptor site and measuring distances from multiple positions (10,(26)(27)(28). Also, as we have shown, regional differences in the binding affinity of the dihistidine site may occur in peptides and proteins.…”

Section: Discussionmentioning

confidence: 99%

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Taraska

Puljung

Zagotta

2009

Proc. Natl. Acad. Sci. U.S.A.

119

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The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports shortrange distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.fluorescence ͉ peptides ͉ FRET F luorescence resonance energy transfer (FRET) occurs when excitation energy is transferred from a donor fluorophore to an acceptor dye through weak dipole-dipole resonance interactions (1, 2). FRET is extremely sensitive to the distance between the two dyes, with the transfer rate inversely proportional to the sixth power of the distance between them. This steep distance dependence has suggested that FRET could be used as a spectroscopic ruler on the molecular scale (3, 4). In addition to distance, five other factors influence the rate of energy transfer: (i) the overlap of the donor's and acceptor's excitation and absorbance spectra, respectively; (ii) the refractive index of the solvent; (iii) the relative orientation of the dyes; (iv) the quantum yield of the donor; and (v) the extinction coefficient of the acceptor.Although FRET is sensitive to the distance between probes, several factors have prevented the widespread use of FRET to easily map backbone distances within proteins. First, most commonly used FRET pairs have R 0 values (distance at which FRET efficiency is 50%) between Ϸ30 and 60 Å and are only able to measure distances between Ϸ20 and 80 Å. Thus, to accurately measure distances, the probes must be separated by large distances relative to the size of many proteins. Also, the large size of many dyes and their long attachment linkers position fluorophores far from the backbone of the protein. These flexible linkers also increase the conformational space sampled by the dye (4, 5). Becau...

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Section: Discussionmentioning

confidence: 99%

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Taraska

Puljung

Zagotta

2009

Proc. Natl. Acad. Sci. U.S.A.

119

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“…To test whether the REC2 domain regulates access of HNH to the target strand scissile phosphate, we labeled SpCas9 with Cy3/Cy5 dyes at positions E60C (within the “stationary” Arginine-rich helix) and D273C (within the “mobile” REC2 domain) to generate SpCas9 REC2 in order to detect REC2 conformational changes (Extended Data Figure 1b–c). We observed reciprocal changes in bulk FRET values ((ratio) A ) 20 between SpCas9 HNH and SpCas9 REC2 across multiple DNA substrates (Extended Data Figure 4g), which suggest that the REC2 and HNH domains are tightly coupled to ensure catalytic competence. smFRET experiments further confirmed a large opening of REC2 during the transition from the sgRNA-bound state (E FRET = 0.96) to the target-bound state (E FRET = 0.43) (Figure 2e–f).…”

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confidence: 87%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

904

819

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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“…Efficiency of energy transfer was reported as (ratio) A whereby the acceptor (AF555) fluorescence via energy transfer is normalized against acceptor fluorescence via direct excitation. (ratio) A parameter is directly proportional to FRET efficiency, and changes in (ratio) A across different experimental conditions serve as a proxy for conformational changes studies (30,35,36). For each FRET construct, a donoronly (FTH-488) sample was prepared and its emission spectrum after 480 nm excitation collected.…”

Section: Fret Analysismentioning

confidence: 99%

“…Indeed, the distance between donor and acceptor dyes can be directly calculated from the FRET efficiency, but accurately relating these variables requires knowledge of numerous complex parameters (36). Without pursuing a more rigorous calculation, we designed a system capable to compare the effect of H-and L-chains in the assembly of the H-chains.…”

Section: Design Of a Fret System Capable To Track Subunit Distances Omentioning

confidence: 99%

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

Carmona

Poli

Bertuzzi

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background: The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H-and Ltype that coassemble in an almost spherical nanocage 12 nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. Methods: We used the Fluorescence Resonance Energy Transfer (FRET) by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. Results: The FRET-efficiency increase from <0.3 of the disassembled to >0.7 in the reassembled allowed to study the self-assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. Conclusion: Heterodimeric (H/L) subunit association is preferred during H/L heteropolymers formation. The H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize and the ferroxidase activity of the heteropolymer reaches a plateau. General significance: This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Keywords

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Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Cited by 79 publications

References 70 publications

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Study of ferritin self-assembly and heteropolymer formation by the use of Fluorescence Resonance Energy Transfer (FRET) technology

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