Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix

Abstract

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

Fig. 1.

PET quenching interactions in polyprolines: steady-state fluorescence, time-resolve fluorescence, and FCS data for F–(Pro)_x–Trp with 0 <x <10 and F being MR121, MR113, and R6G. The number of particles N₀ (filled squares) as detected in FCS are shown compared with static ensemble QY (open squares) for polyprolines labeled with MR113 (a), MR121 (c), and R6G (e). Both curves show good agreement in the appearance of an increasing number of completely quenched, stable complexes with a decreasing length of polyprolines. For MR113 (MR121), the transition midpoint is 3.9 (5.1), and the transition width is 0.3 (2.2). For R6G, a QY minimum is detected for constructs with a single Pro residue. Corresponding data for brightness per particle B (filled circles), as measured by FCS, and the dynamic ensemble QY (open circles) are also in good agreement for labels MR113 (b), MR121 (d), and R6G (e Inset). (f) Normalized FCS data are shown for MR113- (upper curves offset by 1) and MR121-labeled polyprolines (lower curves) and reveal the absence of any fluctuations below the diffusion time of ≈1 ms for all but the following samples: MR113–(Pro)₂–Trp (blue) and MR121–(Pro)_x–Trp with x = 0, 1, or 2 (red, green, blue). However, it should be noted that these fluctuations, as well as dynamic QYs, are detected only for the subpopulations that are not fully quenched because of formation of stable complexes (between 10% and 50% of the total population of the above named samples). The majority of all molecules show no significant fluctuations between 6 ns and the diffusion time ≈1 ms.

Fig. 2.

Prolyl isomerization monitored by changes of PET-quenching efficiency. Steady-state QY for MR121–(Pro)₆–Trp, MR121 (Inset, top curve), MR121–Trp (Inset, bottom curve), and MR121–(GS)₃–Trp (Inset, middle curve) is measured upon solvent exchange from LiCl/TFE (promoting cis isomers) to aqueous solution (promoting trans isomers). The steady-state QY increases exponentially with characteristic time constants on the order of minutes for polyprolines; it remains constant for all control samples that lack a prolyl bond.

Fig. 3.

Point clouds representing conformational degrees of freedom for MR121, MR113, R6G, and Trp attached to polyproline. (a) Structure of MR113–(Pro)₄–Trp with two coordinate systems defined by N and C termini of the polyproline and translated by vector p. Vectors d and t point to the fluorophore and Trp center of mass, respectively. (b and c) Structure of MR121 (b) and R6G (c). All colored bonds represent rotational degrees of freedom. (d–f) Possible orientations of d and t, relative to an all-trans (Pro)₅, are shown for Trp and MR113 (d), MR121 (f), and R6G (g). (e) The influence of a single cis-prolyl isomer on the minimal distance between fluorophore and Trp is shown. All structures are available as a pdb file in supporting information (SI) Appendix.

Fig. 4.

Minimal distances d_min between fluorophore and Trp in polyprolines with a single cis isomer. (a) The conformational space for MR113 and Trp is sampled, the closest approach identified, and d_min is estimated for MR113–(Pro)_x–Trp with x = (2, 3, 4, 5, 6, 8, 10, 12, 15, and 20) (indicated by the length of data curve). All prolyl bonds are in the trans conformation except for a single cis isomer located at the indicated position (counted from the N terminus). The first data point at position 0 represents the all-trans conformation. (b) For each polyproline with x number of residues and a single cis isomer, the configuration that allows closest approach between fluorophore and Trp is identified (i.e., the minimum for each polyproline in a) and d_min displayed. The following structures were investigated with either MR113 (red squares), MR121 (blue circles), or R6G (black triangles) attached: energy-minimized polyproline helix with all bonds in trans conformation (open symbols); energy minimized structures with a single cis bond as shown in a (closed symbols). The black line represents end-to-end distances of a PPII helix as defined from the crystal structure (15) (black line). The dotted lines indicate the distance range above which no quenching interactions are possible.