Imaging Membrane Receptors at Work

The enormous progress in the application of atomic force microscopy (AFM) to imaging, manipulating and parametrizing of biological systems is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Cellular membranes can be imaged in their native state at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Thereby the exceptional signal-to-noise ratio of the AFM does not require to label or fix the biological sample of which single proteins can be clearly observed at sub-nanometer resolution. Because AFM images native biological samples in buffer solution conformational changes that are related to their unique functions can be recorded to a similar resolution, thus complementing the often static structural information acquired by other methods. The unique capability of AFM to directly observe single proteins in their native environments provides insights into their working principles. In addition, single-molecule force spectroscopy (SMFS) combined with single molecule imaging (e.g. force-distance curve-based AFM (FD-based AFM)) provides unprecedented possibilities for observing proteins at work and simultaneously analyzing of their intramolecular and intermolecular forces.

Gap junction channels mediate communication between adjacent epithelial cells. Using AFM, we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native gap junction plaques assembled from connexin 26. The movie shows the extracellular connexon surface imaged before and after injection of Ca(2+) into the buffer solution. In the presence of calcium the extracellular channel entrance reduces its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. The outer diameter of the connexon corresponds to 4.9 nm. The movie was taken from Muller et al. EMBO Journal (2002) 21, 3598-3607.
Since the late 1990ies we apply time-lapse AFM to image membrane proteins at work. The example shows an early work in which we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native gap junction plaques assembled from connexin 26. Gap junction channels mediate communication between adjacent epithelial cells. The movie shows the extracellular connexon surface imaged before and after injection of calcium into the buffer solution. In the presence of calcium the extracellular channel entrance reduces its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. The outer diameter of the connexon corresponds to 4.9 nm. The movie is taken from Muller et al. EMBO Journal (2002) 21, 3598-3607.

Further reading

Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O

E. Mulvihill, K. van Pee, S.A. MariD.J. Müller & Ö. Yildiz
Nano Letters (2015) 15, 6965-6973 external pageonline

Quantitative imaging of the electrostatic field and potential generated by a transmembrane protein at sub-nanometer resolution 

M. Pfreundschuh, U. Hensen & D.J. Muller
Nano Letters (2013) 13, 5585-5593.

Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains

S.A. Mari, J. Pessoa, S.L. Altieri, U. Hensen, L. Thomas, J.H. Morais-Cabral & D.J. Muller
Proc. Natl. Acad. Sci. USA (2011) 108, 20802-20807.

pH induced conformational change of the outer membrane protein OmpG reconstituted into native E. coli lipids

S.A. Mari, C.A. Bippes, S. Köster, Ö. Yildiz, W. Kühlbrandt & D.J. Müller
Journal of Molecular Biology (2010) 396, 610-616.

Aminosulfonate modulated pH induced conformational changes in Connexin26 hemichannels

J. Yu, Ch. Bippes. G. Hand, D.J. Müller & G. Sosinsky,
Journal of Biological Chemistry
(2007) 282, 8895-8904.

Observing membrane protein diffusion at subnanometer resolution

D. J. Müller, A. Engel, Th. Meier, U. Matthey, P. Dimroth & K. Suda
Journal of Molecular Biology
(2003) 327, 925-930.

Conformational changes in surface structures of isolated Connexin26 gap junctions

D.J. Müller, G.M. Hand, A. Engel & G.E. Sosinsky
EMBO Journal
(2002) 21, 3598-3607.

Imaging the electrostatic potential of transmembrane channels: Atomic probe microscopy of OmpF porin

A. Philippsen, W. Im, A. Engel, T. Schirmer, B. Roux & D.J. Müller
Biophysical Journal
(2002) 82, 1667-1676.

Observing proteins at work with the atomic force microscope

A. Engel & D.J. Müller
Nature Structural Biology
(2000) 7, 715-718.

Conformational changes, flexibilities and intramolecular forces observed on individual proteins using AFM

D.J. Müller, D. Fotiadis, C. Möller, S. Scheuring & A. Engel
Single Molecule
(2000) 1, 115-118.

pH and voltage induced structural changes of porin OmpF explains channel closure

D.J. Müller & A. Engel
Journal of Molecular Biology
(1999) 285, 1347-1351.

Structural changes of native membrane proteins monitored at subnanometer resolution with the atomic force microscope

D.J. Müller, C.-A. Schoenenberger, F. Schabert & A. Engel
Journal of Structural Biology
(1997) 119, 149-157.

Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans observed with atomic force microscopy

D.J. Müller, W. Baumeister & A. Engel
Journal of Bacteriology
(1996) 178, 3325-3329.

Force-induced conformational change of bacteriorhodopsin

D.J. Müller, G. Büldt & A. Engel
Journal of Molecular Biology
(1995) 249, 239-243.

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