Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-9-102 s).

Astbury Centre for Structural Molecular Biology University of Leeds Leeds UK

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

School of Biochemistry University of Bristol Bristol UK

School of Clinical and Applied Sciences Faculty of Health and Social Sciences Leeds Beckett University Leeds UK

School of Molecular and Cellular Biology Faculty of Biological Sciences University of Leeds Leeds UK

Zobrazit více v PubMed

Jefferson RE, Min D, Corin K, Wang JY, Bowie JU (2018) Applications of single-molecule methods to membrane protein folding studies. J Mol Biol 430(4):424–437 DOI

Martinac B (2017) Single-molecule FRET studies of ion channels. Prog Biophys Mol Biol 130(Pt B):192–197 DOI

Harborne SPD, Strauss J, Turku A, Watson MA, Tuma R, Harris SA, Goldman A (2018) Defining dynamics of membrane-bound pyrophosphatases by experimental and computational single-molecule FRET. Methods Enzymol 607:93–130 DOI

Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer-Verlag, New York DOI

Calebiro D, Sungkaworn T (2018) Single-molecule imaging of GPCR interactions. Trends Pharmacol Sci 39(2):109–122 DOI

Collinson I, Corey RA, Allen WJ (2015) Channel crossing: how are proteins shipped across the bacterial plasma membrane? Philos Trans R Soc Lond B Biol Sci 370(1679):20150025 DOI

Allen WJ, Corey RA, Oatley P, Sessions RB, Baldwin SA, Radford SE, Tuma R, Collinson I (2016) Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation. Elife 5:e15598 DOI

Corey RA, Allen WJ, Collinson I (2016) Protein translocation: what’s the problem? Biochem Soc Trans 44(3):753–759 DOI

Deville K, Gold VA, Robson A, Whitehouse S, Sessions RB, Baldwin SA, Radford SE, Collinson I (2011) The oligomeric state and arrangement of the active bacterial translocon. J Biol Chem 286(6):4659–4669 DOI

Gold VA, Duong F, Collinson I (2007) Structure and function of the bacterial Sec translocon. Mol Membr Biol 24(5–6):387–394 DOI

Chandradoss SD, Haagsma AC, Lee YK, Hwang JH, Nam JM, Joo C (2014) Surface passivation for single-molecule protein studies. J Vis Exp (86)

Fessl T, Watkins D, Oatley P, Allen WJ, Corey RA, Horne J, Baldwin SA, Radford SE, Collinson I, Tuma R (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination. Elife 7

Sharma A, Leach RN, Gell C, Zhang N, Burrows PC, Shepherd DA, Wigneshweraraj S, Smith DA, Zhang X, Buck M, Stockley PG, Tuma R (2014) Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies. Nucleic Acids Res 42(8):5177–5190 DOI

Preus S, Noer SL, Hildebrandt LL, Gudnason D, Birkedal V (2015) iSMS: single-molecule FRET microscopy software. Nat Methods 12(7):593–594 DOI

Ingargiola A, Laurence T, Boutelle R, Weiss S, Michalet X (2016) Photon-HDF5: an open file format for timestamp-based single-molecule fluorescence experiments. Biophys J 110(1):26–33 DOI

Ingargiola A, Lerner E, Chung S, Weiss S, Michalet X (2016) FRETBursts: an open source toolkit for analysis of freely-diffusing single-molecule FRET. PLoS One 11(8):e0160716 DOI

Hohlbein J, Craggs TD, Cordes T (2014) Alternating-laser excitation: single-molecule FRET and beyond. Chem Soc Rev 43(4):1156–1171 DOI

Torella JP, Holden SJ, Santoso Y, Hohlbein J, Kapanidis AN (2011) Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis. Biophys J 100(6):1568–1577 DOI

Tomov TE, Tsukanov R, Masoud R, Liber M, Plavner N, Nir E (2012) Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis. Biophys J 102(5):1163–1173 DOI

Hoffmann A, Nettels D, Clark J, Borgia A, Radford SE, Clarke J, Schuler B (2011) Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP). Phys Chem Chem Phys 13(5):1857–1871 DOI

Maus M, Cotlet M, Hofkens J, Gensch T, De Schryver FC, Schaffer J, Seidel CA (2001) An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules. Anal Chem 73(9):2078–2086 DOI

Kalinin S, Valeri A, Antonik M, Felekyan S, Seidel CA (2010) Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states. J Phys Chem B 114(23):7983–7995 DOI

Holden SJ, Uphoff S, Hohlbein J, Yadin D, Le Reste L, Britton OJ, Kapanidis AN (2010) Defining the limits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99(9):3102–3111 DOI

Bronson JE, Fei J, Hofman JM, Gonzalez RL Jr, Wiggins CH (2009) Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys J 97(12):3196–3205 DOI

van de Meent JW, Bronson JE, Wiggins CH, Gonzalez RL Jr (2014) Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. Biophys J 106(6):1327–1337 DOI

Konig SL, Hadzic M, Fiorini E, Borner R, Kowerko D, Blanckenhorn WU, Sigel RK (2013) BOBA FRET: bootstrap-based analysis of single-molecule FRET data. PLoS One 8(12):e84157 DOI

McKinney SA, Joo C, Ha T (2006) Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5):1941–1951 DOI

Qin F, Li L (2004) Model-based fitting of single-channel dwell-time distributions. Biophys J 87(3):1657–1671 DOI

Greenfeld M, Pavlichin DS, Mabuchi H, Herschlag D (2012) Single Molecule Analysis Research Tool (SMART): an integrated approach for analyzing single molecule data. PLoS One 7(2):e30024 DOI

Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5(6):507–516 DOI

Li M, Jorgensen SK, McMillan DG, Krzeminski L, Daskalakis NN, Partanen RH, Tutkus M, Tuma R, Stamou D, Hatzakis NS, Jeuken LJ (2015) Single enzyme experiments reveal a long-lifetime proton leak state in a heme-copper oxidase. J Am Chem Soc 137(51):16055–16063 DOI

Fessl T, Lilley DM (2013) Measurement of the change in twist at a helical junction in RNA using the orientation dependence of FRET. Biophys J 105(9):2175–2181 DOI

Blanco M, Walter NG (2010) Analysis of complex single-molecule FRET time trajectories. Methods Enzymol 472:153–178 DOI