Bilayer-Insertion Mechanisms of Self-Inserting Membrane Proteins by Combined Ensemble and Single-Molecule Spectroscopy

• Applicants: Professor Dr. Sandro Keller; Professor Dr. Michael Schlierf
• Subject Area: Biophysics ; Biochemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276454827

• Final Report Year: 2021

Final Report Abstract

Within this project, we have established various methodological advances from time-resolved spectroscopy to single-molecule FRET for membrane protein folding. Exploiting these methods, we have learned many fundamental insights on membrane-protein structure and folding, in particular for the membrane-associated protein Mistic and OmpLA. Morevoer, we exploited our developments also beyond these two model proteins and applied our techniques to obtain fundamental insights into transmembrane a-helix hairpin structures in lipid bilayers, also with regard to disease mutations. Besides our groups, also the field advanced within the past years to study structural dynamics of membrane proteins. This project was key to developing various methodologies, which the Schlierf lab is currently using to study the interaction of outer membrane proteins with periplasmic chaperones and a project funded by the Mukoviszidose e.V. on correction of CFTR transmembrane segment folding with small molecules.

Publications

• 2016. Structural stability of DNA origami nanostructures in the presence of chaotropic agents. Nanoscale 8, 10398-10405
  Ramakrishnan, S., Krainer, G., Grundmeier, G., Schlierf, M., Keller, A.
  (See online at https://doi.org/10.1039/c6nr00835f)
• 2017. Cation-induced stabilization and denaturation of DNA origami nanostructures in urea and guanidinium chloride. Small 13, 1702100
  Ramakrishnan, S., Krainer, G., Grundmeier, G., Schlierf, M., Keller, A.
  (See online at https://doi.org/10.1002/smll.201702100)
• 2017. Slow interconversion in a heterogeneous unfolded-state ensemble of Outer Membrane Phospholipase A. Biophysical Journal 113, 1280-1289
  Krainer, G., Gracia, P., Frotscher, E., Hartmann, A., Gröger, P., Keller, S., Schlierf, M.
  (See online at https://doi.org/10.1016/j.bpj.2017.05.037)
• 2018. A Minimal Helical-Hairpin Motif Provides Molecular-Level Insights into Misfolding and Pharma- cological Rescue of CFTR. Communications Biology 1, 154
  Krainer, G., Treff, A., Hartmann, A., Stone, T., Schenkel, M., Keller, S., Deber, CM., Schlierf, M.
  (See online at https://doi.org/10.1038/s42003-018-0153-0)
• 2018. ATPase and protease domain movements in the bacterial AAA+-protease FtsH are driven by thermal fluctuations. Journal of Molecular Biology 430 (22), 4592-4602
  Ruer, M., Krainer, G., Gröger, P., Schlierf, M.
  (See online at https://doi.org/10.1016/j.jmb.2018.07.023)
• 2018. Conformational Dynamics Govern the Free-Energy Landscape of a Membrane-Interacting Protein. ACS Omega 3 (9), 12026 - 12032
  Frotscher, E., Krainer, G., Hartmann, A., Schlierf, M., Keller, S.
  (See online at https://doi.org/10.1021/acsomega.8b01609)
• 2018. Dissecting Nanosecond Dynamics in a Membrane-Associated Protein Using Dipolar Relaxation Upon Photoexcitation. Journal of Physical Chemistry Letters 9, 2241-2245
  Frotscher, E., Krainer, G., Schlierf, M., Keller, S.
  (See online at https://doi.org/10.1021/acs.jpclett.8b00834)
• 2018. Extracavity effect in cyclodextrin/surfactant complexation. Langmuir 34, 5781-5787
  Vargas, C., Schönbeck, C., Heimann, I., Keller, S.
  (See online at https://doi.org/10.1021/acs.langmuir.8b00682)
• 2018. In situ temperature monitoring in single-molecule FRET experiments. Journal of Chemical Physics 148, 123330
  Hartmann, A., Berndt, F., Ollmann, S., Krainer, G., Schlierf, M.
  (See online at https://doi.org/10.1063/1.5008966)
• 2018. Precision and accuracy of single-molecule FRET measurements—a multilaboratory benchmark study. Nature Methods 15 (9) 669-676
  Hellenkamp, B., Schmid, S., Doroshenko, O., Opanasyuk, O., Kühnemuth, R., Adariani, SR., Barth, A., Birkedal, V., Bowen, ME., Cordes, T., Eilert, T., Fijen, C., Götz, M., Gouridis, G., Ha, T., Hartmann, A., Hendrix, J., Hildebrandt, L., Hohlbein, J., Hübner, CG., Kallis G., Kapanidis, AN., Krainer, G., Lamb, DC., Lemke, EA., Levesquel, B., McCann, JJ., Naredi-Rainer, N., Nettels, D., Qiu, R., Röcker, C., Sanabria, H., Schlierf, M., Schuler, B., Seidel, H., Steit, L., Tinnefeld, P., Tyagi, S., Vandenberk, N., Weninger, KR., Wünsch, B., Yanez- Orozco, IS., Michaelis, J., Seidel, CAM., Craggs, TD., Hugel, T.
  (See online at https://doi.org/10.1038/s41592-018-0085-0)
• 2018. Ultrafast protein folding in membrane-mimetic environments. Journal of Molecular Biology 430 (4), 554-564
  Krainer, G., Hartmann, A., Anandamurugan, A., Gracia, P., Keller, S., Schlierf, M.
  (See online at https://doi.org/10.1016/j.jmb.2017.10.031)
• 2019. Enhancing the stability of DNA origami nanostructures: staple strand redesign versus enzymatic ligation. Nanoscale 11(35), 16270-16276
  Ramakrishnan, S., Schärfen, L., Hunold, K., Fricke, S., Grundmeier, G., Schlierf, M., Keller, A., Krainer, G.
  (See online at https://doi.org/10.1039/c9nr04460d)
• 2019. Structural dynamics of membraneprotein folding from single-molecule. FRET Curr Opin Struct Biol 58, 124-137
  Krainer, G., Keller, S., Schlierf, M.
  (See online at https://doi.org/10.1016/j.sbi.2019.05.025)
• 2020. CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor. JBC 295(7),1985-1991
  Krainer, G., Schenkel, M., Hartmann, A., Ravamehr-Lake, D., Deber, CM., Schlierf, M.
  (See online at https://doi.org/10.1074/jbc.ac119.011360)
• 2020. DIBMA nanodiscs keep a-synuclein folded. Biochmica et Biophysica Acta Biomembranes 1862, 183314-5787
  Adão, R., Cruz, P.F., Vaz, D.C., Fonseca, F., Pedersen, J.N., Ferreira-da-Silva, F., Brito, R.M.M., Ramos, C.H.I., Otzen, D., Keller, S., Bastos, M.
  (See online at https://doi.org/10.1016/j.bbamem.2020.183314)
• 2020. SDS-induced multi-state unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence. Chemical Science 11, 9141-9153
  Krainer, G., Hartmann, A. , Bogatyr, V. , Nielsen, J., Schlierf, M., Otzen, D.
  (See online at https://doi.org/10.1039/d0sc02100h)
• 2020. Towards next generation therapies for cystic fibrosis: Folding, function and pharmacology of CFTR. Journal of Cystic Fibrosis 19, S25-S32
  Bose, SJ., Krainer, G., Ng DRS., Schenkel, M., Shishido, H., Yoon JS., Haggie, PM., Schlierf, M., Sheppard, DN., Skach, WR.
  (See online at https://doi.org/10.1016/j.jcf.2019.12.009)