Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence

  • To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin moleculaTo determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances.show moreshow less

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Metadaten
Author:Benjamin Schuler, Everett A. Lipman, Peter J. Steinbach, Michael Kumke, William A. Eaton
URN:urn:nbn:de:kobv:517-opus-12229
Series (Serial Number):Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, ISSN 1866-8372 (paper 008)
Document Type:Postprint
Language:English
Date of Publication (online):2007/01/26
Year of Completion:2005
Publishing Institution:Universität Potsdam
Release Date:2007/01/26
Tag:FRET; Förster resonance energy transfer; molecular dynamics; polypeptide
Source:Proceedings of the National Academy of Scienes of the United States of America. - ISSN 1091-6490. -102 (2005), 8, S. 2754 - 2759
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Collections:Universität Potsdam / Aufsätze (Pre- und Postprints) / Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie / Physikalische Chemie
Notes extern:
first published in:
Proceedings of the National Academy of Scienes of the United States of America - ISSN 1091-6490. - 102 (2005), S. 2754 - 2759
doi: 10.1073/pnas.0408164102