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

Abstract

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.

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

“…DFRET is just one method in a whole range of optical methods for studying the structure and dynamics of polymers [ 10 , 11 , 12 , 13 , 14 , 17 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. When we go into detail, we realize that any progress in DFRET can synergistically promote progress in this entire field [ 13 ].…”

“…We established that DI increases with J and that J increases linearly with D and τ D , the donor lifetime, and decreases with the square root of the Förster radius, R 0 (Equation (18)). All of these variations have been employed in earlier attempts at a global analysis but without knowing which range of the diffusion influence was actually covered [ 3 , 7 , 9 , 10 , 14 ]. In this work, we obtained certainty that our previous variation [ 14 ] of the diffusion coefficient by solvent viscosity and variation of the donor lifetime by adding the viscogen ethylene glycol and by going from the donor FTrp to the donor NAla covered a DI range of 8 to 80% (comp.…”

“…We can therefore use the HSE to globally fit the experimental data. This was quite arduous in the past, where the HSE served a bit as a black box [ 5 , 6 , 10 , 11 , 12 , 13 , 14 ]. In the Results Section, we develop the Haas-Steinberg–Jacob equation (HSJE), a closed analytical expression (CAE).…”

“…There is also no question about the significance of Förster resonance energy transfer in structural and dynamic molecular studies as the number of citations to Förster’s original paper approaches 10,000 [ 2 ]. We believe that diffusion-enhanced fluorescence resonance energy transfer, or DFRET, on the molecule ensemble can also develop into an influential method but is, as of now, difficult to handle and non-transparent in terms of data acquisition and analytics [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. This article aims to make DFRET accessible to any scientist working in one of the many fields of polymer structure and dynamics.…”

“…The story of diffusion-enhanced FRET or DFRET started 40 years ago [ 3 ]. Since then, the number of published articles based on DFRET has remained small, not for lack of interest but for the method’s complexity [ 4 , 5 , 6 , 8 , 10 , 11 , 12 , 13 , 14 ]. In 1978, Steinberg derived what we began to call the Haas-Steinberg equation (HSE) [ 3 ].…”