title: In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.
abstract: Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems, Molecular Biophysics (ICS-5), 52425 Jülich, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Baumann
authors.affiliation: Freie Universität Berlin, Department of Physics, Experimental Molecular Biophysics, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Kerruth
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems, Molecular Biophysics (ICS-5), 52425 Jülich, Germany.|Physikalisches Institut (IA), AG Biophysik, RWTH Aachen, Sommerfeldstrasse 14, 52074 Aachen, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fitter
authors.affiliation: Forschungszentrum Jülich, Institute of Complex Systems, Molecular Biophysics (ICS-5), 52425 Jülich, Germany.|Moscow Institute of Physics and Technology, Laboratory for Advanced Studies of Membrane Proteins, 141700 Dolgoprudniy, Russia.
authors.forename: Georg
authors.initials: G
authors.lastname: Büldt
authors.affiliation: Freie Universität Berlin, Department of Physics, Experimental Molecular Biophysics, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
authors.affiliation: Freie Universität Berlin, Department of Physics, Genetic Biophysics, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Ramona
authors.initials: R
authors.lastname: Schlesinger
authors.affiliation: Freie Universität Berlin, Department of Physics, Experimental Molecular Biophysics, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ataka
mesh_terms: D002474:Cell-Free System; D008565:Membrane Proteins / Q000737:chemistry*; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D008565:Membrane Proteins