Short‐Distance FRET Applied to the Polypeptide Chain

Jacob, Maik H.; Nau, Werner M.

doi:10.1002/9781118183373.ch4

Cited by 3 publications

(6 citation statements)

References 67 publications

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“…The peptide, NAla-(GS) 6 -Dbo-NH 2 , shows a fluorescence spectrum (red curve) with two regions of maximal intensity, one around 340 nm arising from NAla fluorescence and another at 440 nm due to Dbo fluorescence. Since Dbo in the absence of a donor cannot be optically excited at 280 nm [12], it receives its excitation from NAla through FRET. In the presence of increasing concentrations of ethylene glycol, the fluorescence from NAla increases and the fluorescence from Dbo decreases.…”

Section: Resultsmentioning

confidence: 99%

“…Large Förster radii are suitable to detect large distances; small radii are required if short distances and minute distance differences have to be detected. We introduced short-distance FRET (sdFRET), where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as an acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-fluorotryptophan, or tyrosine [10,11,12,13]. The Förster radii are always about 10 Å, which is about half of the lower limit offered by traditional donor-acceptor pairs.…”

Section: Introductionmentioning

confidence: 99%

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Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

et al. 2018

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A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods (R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

et al. 2018

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“…The difference in probe–probe interactions of the systems in PET and TTET can readily account for the factor of 2 difference in quenching rates between PET and TTET. It can also explain the ratio of about 2.6 between rates from TTET and Trp CIFQ as the Dbo/Trp pair is least prone to hydrophobic interactions because the DBO fluorophore is hydrophilic …”

Section: Discussionmentioning

confidence: 99%

“…It can also explain the ratio of about 2.6 between rates from TTET and Trp CIFQ as the Dbo/Trp pair is least prone to hydrophobic interactions because the DBO fluorophore is hydrophilic. 36 Complex formation between hydrophobic probes can be seen as a model for the loop node. Thus, loop rates will also depend on and inform changes in the energy landscape caused by the loop-node constituting residues.…”

Section: ■ Discussionmentioning

confidence: 99%

“…With the idea to establish upper limits, beyond which, as we initially thought, rate values could not be realistic, we have applied a complementary fluorescence-based method, namely, short-distance Förster resonance energy transfer (sdFRET), − and compare herein the sdFRET quenching rates with the loop rates derived from TTET, PET, and CIFQ. Generally, FRET rates are expected to exceed loop rates in identical chains: a single energy-transfer event can occur at a distance but becomes almost inevitable when probes can approach each other within the natural fluorescence lifetime of the donor, a phenomenon known as FRET diffusion enhancement. ,, We benefited from the fact that Dbo combined with either Trp, FTrp, or NAla enables not only CIFQ but also sdFRET, through which distances of around 10 Å have recently become accessible to FRET detection.…”

Section: Introductionmentioning

confidence: 99%

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Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding

et al. 2018

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Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.

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Diffusion-Enhanced Förster Resonance Energy Transfer in Flexible Peptides: From the Haas-Steinberg Partial Differential Equation to a Closed Analytical Expression

et al. 2023

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In the huge field of polymer structure and dynamics, including intrinsically disordered peptides, protein folding, and enzyme activity, many questions remain that cannot be answered by methodology based on artificial intelligence, X-ray, or NMR spectroscopy but maybe by fluorescence spectroscopy. The theory of Förster resonance energy transfer (FRET) describes how an optically excited fluorophore transfers its excitation energy through space to an acceptor moiety—with a rate that depends on the distance between donor and acceptor. When the donor and acceptor moiety are conjugated to different sites of a flexible peptide chain or any other linear polymer, the pair could in principle report on chain structure and dynamics, on the site-to-site distance distribution, and on the diffusion coefficient of mutual site-to-site motion of the peptide chain. However, the dependence of FRET on distance distribution and diffusion is not defined by a closed analytical expression but by a partial differential equation (PDE), by the Haas-Steinberg equation (HSE), which can only be solved by time-consuming numerical methods. As a second complication, time-resolved FRET measurements have thus far been deemed necessary. As a third complication, the evaluation requires a computationally demanding but indispensable global analysis of an extended experimental data set. These requirements have made the method accessible to only a few experts. Here, we show how the Haas-Steinberg equation leads to a closed analytical expression (CAE), the Haas-Steinberg-Jacob equation (HSJE), which relates a diffusion-diagnosing parameter, the effective donor–acceptor distance, to the augmented diffusion coefficient, J, composed of the diffusion coefficient, D, and the photophysical parameters that characterize the used FRET method. The effective donor–acceptor distance is easily retrieved either through time-resolved or steady-state fluorescence measurements. Any global fit can now be performed in seconds and minimizes the sum-of-square difference between the experimental values of the effective distance and the values obtained from the HSJE. In summary, the HSJE can give a decisive advantage in applying the speed and sensitivity of FRET spectroscopy to standing questions of polymer structure and dynamics.

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Short‐Distance FRET Applied to the Polypeptide Chain

Cited by 3 publications

References 67 publications

Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding

Diffusion-Enhanced Förster Resonance Energy Transfer in Flexible Peptides: From the Haas-Steinberg Partial Differential Equation to a Closed Analytical Expression

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