Characterization of the motion of membrane proteins using high-speed atomic force microscopy

Journal name:
Nature Nanotechnology
Volume:
7,
Pages:
525–529
Year published:
DOI:
doi:10.1038/nnano.2012.109
Received
Accepted
Published online

Abstract

For cells to function properly1, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein–protein and protein–lipid interactions2. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy3. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein–protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

At a glance

Figures

  1. HS-AFM movie frames showing the motion of OmpF trimers in the membrane.
    Figure 1: HS-AFM movie frames showing the motion of OmpF trimers in the membrane.

    Full movie length, 115,434 ms; frame rate, 477 ms; full image size, 750 Å; full frame size, 300 pixels (Supplementary Movie S1). a, Individual HS-AFM frames. The individual subunits of the OmpF trimers are resolved. b, As in a, but with an overlaid outline of OmpF trimer localization and orientation. c, Molecular positions in the current frame (red outlines) compared with those of the previous frame (blue outlines) with displacement shown as black lines. d, Molecular displacement traces and orientations (arrows) as analysed in c. Note that the angular orientation depicts one subunit of the trimeric porin OmpF. Black crosses and bars display the average and standard deviation of the position of the molecules. The network overlay presents the Voronoi tessellation defining the free membrane area of each molecule.

  2. Diffusion analysis of OmpF.
    Figure 2: Diffusion analysis of OmpF.

    a, Histogram of the displacement velocity of the molecules: blue, representative immobile molecule participating in a stable association; red, representative diffusing molecule. b,c, Diffusion traces (b) and MSD as a function of lag time (c) of the two molecules. The derived exponent values (fit ± s.d.) are α = 0.1 ± 0.1 and α = 1.1 ± 0.2 for the immobile and diffusing molecule, respectively.

  3. MDS and experimental evaluation of OmpF-OmpF interaction pathway and potential.
    Figure 3: MDS and experimental evaluation of OmpF–OmpF interaction pathway and potential.

    a, Potential energy landscape from coarse-grained MDS. Top: interaction energy between two molecules in the membrane as a function of distance with a shortest contact association well at a centre-to-centre distance of 7 nm and a repulsive regime at separations further than 9 nm. The trajectory traverses ‘base-to-base’, ‘tip-to-base’ and ‘tip-to-tip’ configurations at distances of 7.0 nm, 7.5 nm and 8.5 nm, respectively, until loss of molecular contact at 9.5 nm. Asterisks indicate high-energy intermediate configurations that are probably under-sampled. Bottom: umbrella sampling histograms used to construct the potential. b, The separation of two OmpF proteins implies molecular shear and rotational movements. The observed transition comprises all of the major interaction states that are low-energy states in the coarse-grained MDS shown in a. The two molecules are initially in a ‘base-to-base’ association (477 ms), then, via a rotational rearrangement, they translate into a ‘tip-to-tip’ configuration (1,908 ms); further rotation brings them back into a ‘base-to-tip’ association (3,339 ms), before engaging again in a ‘tip-to-tip’ association (5,247 ms, 5,724 ms) then separation (6,201 ms). The two central molecules are identified within yellow dashed lines, and triangle symbols in the top left corner represent their interaction state (white triangles represent loss of contact as in a). Both molecules engage in relationships with neighbouring molecules (6,678 ms, 7,155 ms) (Supplementary Movie S6). c, Experimental interaction probability map: a multidimensional representation, where the radial distance is the centre-to-centre distance (r, nm) between two molecules, the polar coordinate describes the localization of the neighbouring molecule with respect to the central molecule, the colour coding on the vertical dimension represents the relative orientation between the two molecules, with pairs of triangle symbols (as in a and b) on the densities indicating the respective orientation of trimers occurring with high probability. The three-dimensional density surface is set at fivefold enrichment relative to the mean probability.

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Author information

Affiliations

  1. U1006 INSERM, Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009 Marseille, France

    • Ignacio Casuso,
    • Mohamed Husain &
    • Simon Scheuring
  2. UPR-9027 LISM, CNRS-Aix-Marseille University, Marseille, 13402, France

    • Jonathan Khao,
    • Jean-Pierre Duneau &
    • James N. Sturgis
  3. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University Basel, Mattenstrasse 26, WRO-1058, CH-4058 Basel, Switzerland

    • Mohamed Chami &
    • Henning Stahlberg
  4. Cell and Tissue Imaging Facility (PICT-IBiSA), UMR144 CNRS-Institut Curie, 26 rue d'Ulm, Paris, F-75248 France

    • Perrine Paul-Gilloteaux

Contributions

I.C. and S.S. conceived the experiments. I.C., M.H., M.C. and J.K. performed experiments. I.C., S.S., M.H., P.P-G., J-P.D. and J.N.S. analysed the data. I.C., S.S., J.N.S. and H.S. co-wrote the paper.

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The authors declare no competing financial interests.

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