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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.

Biopolymers, such as single-stranded DNA (ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA (polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length Lp, we found that Lp approximately Im, with m=-0.22+/-0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics (MD) simulations with a force field that accounts for short-range interactions in vacuum, and determined the characteristic polymer properties end-to-end distance R, radius of gyration S, and persistence length Lp of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental Rh measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with >approximately 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of approximately 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.