HIGH-RESOLUTION ATOMIC FORCE MICROSCOPY OF NATIVE MEMBRANES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 24

interest were initially isolated from biological membranes, followed by 2D
reconstitution to form regular arrangements or dense packing. However,
the shortcoming of this procedure is that the isolation and reconstitution
processes may disturb the native state of membrane proteins. Understanding
the function of a membrane protein requires its structural study in the native
membrane; additionally, this allows the protein of interest to be observed
together with other partner proteins.
In addition to high-resolution topographic imaging, another key advantage
of AFM is its excellent capacity to nano-manipulate individual membrane
proteins by applying additional loading forces to the imaging tip, along
with adapted scan rates and feedback parameters to deliberately act on the
surface of the biological object ( Fig. 2.3a,d ) . When additional loading forces
are relatively high, stacked membrane layers can be dissected to give access
to underlying membranes. Moreover, individual protein subunits of multi-

protein complexes can be dissected at slightly increased forces, allowing for
the analysis of underlying protein structures. 17,23,24 At low additional loading
forces, individual protein domains can be manipulated. This process is non-
destructive and provides access to the analysis of lexible protein surface
motifs.

18-22

High-resolution imaging and manipulation often beneit each
other during the study of membrane protein structure.

3,22,25

2.2.1 Surface Layers

Surface layers (S-layers) are regular, 2D protein networks, functioning
as the outermost cell wall layer of many bacteria and archaea. 26,27 These
layers withstand non-physiological pH, radiation, temperature, proteolysis,
pressure and detergent treatment, thus protecting the cell from such hostile
factors.

28,29
PS2 is the protein that forms the S-layer of Corynebacterium glutamicum. 30,31

Native S-layers stick together via their inner surfaces in

aqueous condition, with the outer surfaces exposed on opposite sides ( Fig.
2.3a ). When imaging at a minimal loading force of 100 pN, the outer S-layer
surface of the top layer is imaged at high resolution, showing triangle-shaped
protrusions with unit cell dimensions of

C. glutamicum

= 60 ±
1° ( Fig. 2.3b ) . 20 The hexameric core between the triangles could be identiied
as the other side of the lower-shaped hexameric core visible on the inner
surface. During image acquisition, the loading force to the tip is increased
to 500 pN. This mechanical treatment punctures the top S-layer and gave
the AFM tip access to the inner surface of the layer below without severe
damage, exposing a lower-shaped surface ( Fig. 2.3c ). A 3D reconstruction
of the S-layer architecture has been calculated from the topographies of both
surfaces.

a = b = 16.0 ± 0.2 nm and γ

20