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

doi:10.1073/pnas.0705605104

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

Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany

Edited by Harold A. Scheraga, Cornell University, Ithaca, NY, and approved September 14, 2007 (received for review June 15, 2007)

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.

Footnotes

*To whom correspondence may be addressed. E-mail: sdoose{at}physik.uni-bielefeld.de or sauer{at}physik.uni-bielefeld.de
Author contributions: S.D. and H.N. contributed equally to this work; S.D., H.N., and M.S. designed research; S.D., H.N., and H.B. performed research; S.D., H.N., and H.B. analyzed data; and S.D., H.N., and M.S. wrote the paper.
↵ ^†Present address: Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0705605104/DC1.
Abbreviations:

F,

fluorophore;

FCS,

fluorescence correlation spectroscopy;

FRET,

Förster resonance energy transfer;

NHS,

N-hydroxysuccinimidyl;

PET,

photoinduced electron transfer;

PPII,

polyproline type II;

QY,

quantum yield.
Freely available online through the PNAS open access option.