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 Fö rster 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-Lprolines 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.fluorescence correlation spectroscopy ͉ molecular ruler ͉ prolyl isomerization ͉ biopolymer ͉ Förster resonance energy transfer F luorescence spectroscopy provides a variety of tools to measure distances, monitor dynamics, or observe molecular interactions with sensitivity far beyond that of other biophysical techniques. Distance-dependent interactions between fluorophores and fluorescence-quenching molecular compounds, such as Förster resonance energy transfer (FRET) or photoinduced electron transfer (PET), are capable of reporting on processes on the nanometer length scale, below the diffraction limit generally imposed on optical techniques. Although much information is gained from relative distances or distance changes, the quest for precise determination of absolute distances has been ongoing ever since Stryer and Haugland presented the application of FRET as a spectroscopic ruler (1). FRET is a nonradiative dipole-dipole interaction between two fluorophores (donor D and acceptor A) that can serve as a spectroscopic ruler on length scales between Ϸ2 and Ϸ10 nm (2-6). Structurally well defined model systems (molecular rulers) are needed for validation of spectroscopic rulers as much as the latter are essential for structural investigations. Since the sem...

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer

“…Although indication for PPII structure was observed (10), the end-to-end distance of p53N62-78 and p53N74-92 was significantly shorter than expected from ideal PPII structure [similar to results from SM-FRET data of p53N(56C91C)]. The existence of cis-proline (34,35) and flexible residues (36) may significantly decrease the end-to-end distance observed by FRET. A compacted conformation of PPII-forming peptides is also observed with other techniques (37).…”

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

“…[8] This discrepancy could recently be ascribed to cis-trans isomerization of individual proline residues leading to a deviation from the expected stiff structure. [3,9,10] FRET as well as intramolecular quenching experiments revealed that a significant fraction of polyproline residues adopted a conformation that involved much shorter distances between donor and acceptor under relevant conditions. Due to the simplicity to produce donor-and acceptor-labeled double-stranded (ds) DNA, oligonucleotides have been used most frequently and FRET was used to illustrate the helical structure of DNA.…”

Conformations and free energy landscapes of polyproline peptides

“…However, both FRET and photoinduced-electron transfer (PET) studies show deviations of experimentally observed end-to-end distances of polyproline from theoretical predictions. Recently, Doose et al used PET techniques to probe the structure and dynamics of PPII with 1-10 residues in aqueous solution (43). The authors showed that "polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-toend distances that do not interconvert on the scales from nano-to milliseconds".…”