Category: accepted

Published: Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study

B. Hellenkamp, S. Schmid, O. Doroshenko, O. Opanasyuk, R. Kühnemuth, S. Rezaei Adariani, B. Ambrose, M. Aznauryan, A. Barth, V. Birkedal, M.E. Bowen, H. Chen, T. Cordes, T. Eilert, C. Fijen, C. Gebhardt, M. Götz, G. Gouridis, E. Gratton, T. Ha, P. Hao, C.A. Hanke, A. Hartmann, J. Hendrix, L.L. Hildebrandt, V. Hirschfeld, J. Hohlbein, B.g Hua, C.G. Hübner, E. Kallis, A.N. Kapanidis, J.Y. Kim, G. Krainer, D.C. Lamb, N.K. Lee, E.A. Lemke, B. Levesque, M. Levitus, J.J. McCann, N. Naredi-Rainer, D. Nettels, T. Ngo, R. Qiu, N.C. Robb, C. Röcker, H. Sanabria, M. Schlierf, T. Schröder, B. Schuler, H. Seidel, L. Streit, J. Thurn, P. Tinnefeld, S. Tyagi, N. Vandenberk, A. Manuel Vera, K.R. Weninger, B. Wünsch, I.S. Yanez-Orozco, J. Michaelis, C.A.M. Seidel, T.D. Craggs, T. Hugel, Nature Methods, 15, 669, 2018, [link], preprint on arXiv: [link]

Single-molecule Förster resonance energytransfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ± 0.02 and ± 0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Publication: Probing the conformational landscape of DNA polymerases using diffusion-based single-molecule FRET

J. Hohlbein and A.N. Kapanidis, Methods in Enzymology: Single-molecule Enzymology Part A & B, 581, 353-378, 2016, [link]

Monitoring conformationalchanges in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open and the closed conformations of DNA polymerases, our smFRET assays utilising doubly labelled variants of E. coli DNA polymerase I were pivotal in identifying and characterising a partially-closed conformation as a primary checkpoint for nucleotide selection. Here, we provide a comprehensive overview of the methods we used for the conformational analysis of wild-type DNA polymerase and some of its low-fidelity derivatives; these methods include strategies for protein labelling and our procedures for solution-based single-molecule fluorescence data acquisition and data analysis. We also discuss alternative single-molecule fluorescence strategies for analysing the conformations of DNA polymerases in vitro and in vivo.

2016_Hohlbein_CH0030_Fig003_Kapanidis_v1_Orig

Publication: Single molecule 3D orientation in Time and Space: A 6D dynamic study on fluorescent labeled lipid membranes.

R. Börner, N. Ehrlich, J. Hohlbein, C.G. Hübner, Journal of Fluorescence, 26, 963-975, 2016 [link]

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation to each other. Recently, we demonstrated a technique that allows the orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As tCapture2he method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, thus, the in cooperation of lipid dyes (DiO) represents a a wide range of dipole orientations. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach.