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Probing Polyproline Structure and Dynamics by Photoinduced Electron Transfer Provides Evidence for Deviations from a Regular Polyproline Type II Helix

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Probing Polyproline Structure and Dynamics by Photoinduced Electron Transfer Provides Evidence for Deviations from a Regular Polyproline Type II Helix Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix So¨ ren Doose*, Hannes Neuweiler†, Hannes Barsch, and Markus Sauer* Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, Universita¨tsstrasse 25, 33615 Bielefeld, Germany Edited by Harold A. Scheraga, Cornell University, Ithaca, NY, and approved September 14, 2007 (received for review June 15, 2007) Polyprolines are well known for adopting a regular polyproline Proline is unique among the natural amino acids in having a side type II helix in aqueous solution, rendering them a popular stan- chain that is cyclized to the backbone, restricting its conformational dard as molecular ruler in structural molecular biology. However, space considerably (12). Polyprolines have been characterized by single-molecule spectroscopy studies based on Fo¨rster resonance various spectroscopic techniques (1, 7, 8, 13–21). Two main con- energy transfer (FRET) have revealed deviations of experimentally formations depending on the isomerization state of the prolyl bond observed end-to-end distances of polyprolines from theoretical were identified: the polyproline type I helix (PPI) with all peptide predictions, and it was proposed that the discrepancy resulted bonds in the cis conformation (14); and the polyproline type II helix from dynamic flexibility of the polyproline helix. Here, we probe (PPII) with all peptide bonds being trans isomers (15, 16). However, end-to-end distances and conformational dynamics of poly-L- the notion that polyprolines constitute a system as well defined in prolines with 1–10 residues using fluorescence quenching by structure as suggested by crystallography has been questioned. photoinduced-electron transfer (PET). A single fluorophore and a Whereas most studies suggested that polyprolines with Ͼ3–5 resi- tryptophan residue, introduced at the termini of polyproline pep- dues form a PPII structure in aqueous solution (19), experimental tides, serve as sensitive probes for distance changes on the sub- evidence for significant deviations from this ideal structure was nanometer length scale. Using a combination of ensemble fluo- found by ensemble FRET (22), single-molecule FRET (7, 8, 23), rescence and fluorescence correlation spectroscopy, we and NMR studies (13, 20). Inspired by molecular dynamics simu- demonstrate that polyproline samples exhibit static structural lations, it was suggested that the PPII structure of polyprolines heterogeneity with subpopulations of distinct end-to-end dis- resembles a continuously bend worm-like chain with a persistence tances that do not interconvert on time scales from nano- to length, i.e., the length on which significant bending becomes milliseconds. By observing prolyl isomerization through changes in appreciable, smaller than thought previously (8, 22). On the other PET quenching interactions, we provide experimental evidence hand, theoretical studies predicted reduced end-to-end distances that the observed heterogeneity can be explained by interspersed caused by small amounts of interspersed cis isomers (24, 25). cis isomers. Computer simulations elucidate the influence of trans/ It is known that the amino acid tryptophan (Trp) can effi- cis isomerization on polyproline structures in terms of end-to-end ciently quench the emission of some strongly fluorescent oxazine distance and provide a structural justification for the experimen- and rhodamine derivatives either through transient collision or tally observed effects. Our results demonstrate that structural by formation of a nonfluorescent complex that is stabilized by heterogeneity inherent in polyprolines, which to date are com- hydrophobic interactions (26, 27). The contact-induced quench- monly applied as a molecular ruler, disqualifies them as appropri- ing process based on PET requires overlap of fluorophore and ate tool for an accurate determination of absolute distances at a quencher molecular orbitals, i.e., van der Waals contact, and molecular scale. thus is sensitive to subnanometer distances (28). Because PET can effectively switch off fluorescence only upon van der Waals fluorescence correlation spectroscopy ͉ molecular ruler ͉ contact between fluorophore and Trp, it can be used as a prolyl isomerization ͉ biopolymer ͉ Förster resonance energy transfer measuring rod probing whether the distance between the two moieties is above or below a critical distance. Related dynamics luorescence spectroscopy provides a variety of tools to mea- down to nanosecond time scales can be investigated by analyzing Fsure distances, monitor dynamics, or observe molecular PET interactions using fluorescence correlation spectroscopy interactions with sensitivity far beyond that of other biophysical (PET–FCS) (27, 29–31). techniques. Distance-dependent interactions between fluoro- In this work we investigate poly-L-prolines by using contact- phores and fluorescence-quenching molecular compounds, such induced PET between a site-specifically attached fluorophore (F) as Fo¨rster 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 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 imposed on optical techniques. Although much information is data; and S.D., H.N., and M.S. wrote the paper. gained from relative distances or distance changes, the quest for The authors declare no conflict of interest. precise determination of absolute distances has been ongoing This article is a PNAS Direct Submission. ever since Stryer and Haugland presented the application of FRET as a spectroscopic ruler (1). FRET is a nonradiative Freely available online through the PNAS open access option. dipole–dipole interaction between two fluorophores (donor D Abbreviations: F, fluorophore; FCS, fluorescence correlation spectroscopy; FRET, Fo¨rster resonance energy transfer; NHS, N-hydroxysuccinimidyl; PET, photoinduced electron trans- and acceptor A) that can serve as a spectroscopic ruler on length fer; PPII, polyproline type II; QY, quantum yield. Ϸ Ϸ scales between 2 and 10 nm (2–6). Structurally well defined *To whom correspondence may be addressed. E-mail: [email protected] or model systems (molecular rulers) are needed for validation of [email protected]. spectroscopic rulers as much as the latter are essential for †Present address: Medical Research Council Centre for Protein Engineering, Hills Road, structural investigations. Since the seminal work of Stryer and Cambridge CB2 0QH, United Kingdom. Haugland with polyprolines as a molecular ruler, both polypep- This article contains supporting information online at www.pnas.org/cgi/content/full/ tides and polynucleotides with well defined structures were used 0705605104/DC1. to validate Fo¨rster theory (4, 7–11). © 2007 by The National Academy of Sciences of the USA 17400–17405 ͉ PNAS ͉ October 30, 2007 ͉ vol. 104 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0705605104 Downloaded by guest on October 2, 2021 and the amino acid Trp: F–(Pro)x–Trp with 0 Ͻx Ͻ10. Computa- tional models of polyproline assuming a regular PPII helix dem- onstrate that for x Ͼ6 residues, fluorescence-quenching contact formation between Trp and fluorophore is not possible because of steric separation. In fluorescence experiments, however, we find significant quenching even for constructs where fluorophore and Trp are spaced by Ͼ6 residues. To investigate conformational dynamics of polyproline peptides, we analyzed the time evolution of fluorescence quenching by time-resolved spectroscopy and FCS experiments. We found that fluorescence-quenched chain config- urations are essentially nonfluorescent on time scales from nano- to milliseconds. Furthermore, we observed considerable steady-state fluorescence changes upon induced prolyl cis/trans isomerization. The observed effects are in agreement with computational models of end-to-end distances of a PPII helix distorted by cis-prolyl isomers. Results and Discussion Steady-State Fluorescence Quenching Experiments of Polyproline Conjugates. We investigated F–(Pro)x–Trp conjugates with three different fluorophores F (MR121, MR113, and R6G) and 0 Ͻx Ͻ10 by using steady-state fluorescence spectroscopy, time- resolved spectroscopy, and FCS (Fig. 1). Steady-state fluores- cence measurements revealed a strong dependence of the steady-state quantum yield (QYss, calculated from the fluores- cence emission I of the quenched molecule relative to I0 of a nonquenched control sample) on the number of proline residues (Fig. 1). From investigating bimolecular interactions between BIOPHYSICS Trp and various fluorophores, we know that QYss is reduced through static and dynamic quenching processes based on PET between fluorophore and Trp. Static quenching describes for- Fig. 1. PET quenching interactions in polyprolines: steady-state fluores- Ͻ Ͻ mation of efficiently quenched complexes in which fluorophore cence, time-resolve fluorescence, and FCS data for F–(Pro)x–Trp with 0 x 10 and Trp favor a stacked configuration that is stabilized by and F being MR121, MR113, and R6G. The number of particles N0 (filled hydrophobic interactions (32). Dynamic quenching takes place squares) as detected in FCS are shown compared with static ensemble QY (open squares) for polyprolines labeled with
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