Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

Abstract

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Fö rster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water Ϸ30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9 -13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R 0 required to fit the data in the classic study of Stryer and Haugland.fluorescence ͉ molecular dynamics ͉ persistence length ͉ polypeptide ͉ proteins F örster resonance energy transfer (FRET) has been a widely used tool for determining distances within and between biological molecules, and was called a ''spectroscopic ruler'' by Stryer and Haugland (1, 2). The basis of the technique is that the rate of energy transfer, k ET , between two chromophores depends on their separation in space R according to k ET ϭ k D (R 0 /R) 6 , as shown by Förster (k D Ϫ1 is the fluorescence lifetime of the ''donor'' chromophore and R 0 the distance at 50% transfer efficiency). The prediction of an R Ϫ6 dependence of k ET was initially confirmed in the experiment by Stryer and Haugland (1), who used a series of L-proline oligomers (Fig. 1A) as spacers of known length (3) between naphthyl and dansyl chromophores. Based on the early theoretical estimate by Schimmel and Flory (4) of 22 nm for the persistence length of poly-L-proline in the type II conformation, the spacers of 1-12 prolines were considered as rigid rods, resulting in a good fit of the theory to the experimental transfer efficiencies. An unexplained result, however, was that the fit required an R 0 significantly longer than the value determined spectroscopically.Advances in detector technology and the use of fluorophores with very high extinction coefficients, large quantum yields, and photochemical sta...

Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories

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Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

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Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

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