High-resolution AFM-imaging and mechanistic analysis of the 20 S proteasome.

As macromolecular protease complex, the 20 S proteasome is responsible for the degradation of cellular proteins and the generation of peptide epitopes for antigen presentation. Here, structural and functional aspects of the 20 S proteasome from Thermoplasma acidophilum have been investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR). Due to engineered histidine tags introduced at defined positions, the proteasome complex was pre-oriented at ultra-flat chelator lipid membranes allowing for high-resolution imaging by AFM. Within these two-dimensional protein arrays, the overall structure of the proteasome and the organization of individual subunits was resolved under native conditions without fixation or crosslinking. In addition, the substrate-proteasome interaction was monitored in real-time by SPR using a novel approach. Instead of following enzyme activity by product formation, the association and dissociation kinetics of the substrate-proteasome complex were analyzed during proteolysis of the polypeptide chain. By blocking the active sites with a specific inhibitor, the substrate binding step could be dissected from the degradation step thus resolving mechanistic details of substrate recognition and cleavage by the 20 S proteasome.

Atomic-force microscopy on the olfactory dendrites of the silkmoths antheraea polyphemus and A. pernyi

Olfactory transduction is thought to occur in the outer dendritic membrane of insect olfactory receptor neurons. Electrophysiological studies have indicated that the outer dendritic membrane has non-specific cation channels and inositol-triphosphate-dependent Ca2+ channels. The presence of such channels is further supported by the observation that pheromone-stimulated dendrites take up cobalt. However, to date, there is no structural evidence for these channels. Therefore, in order to search for putative ion channels, we have imaged the membrane of the olfactory dendrites in the scanning electron microscope (SEM) and the atomic-force microscope (AFM), after extruding the dendrites out of the olfactory hairs and fixing them on plastic coverslips. With the aid of the SEM, we could see the beaded structure of the dendrite but no fine structural details, as the membrane was sputtered with gold. With the use of the contact mode of the AFM, we could see "pores" that were deeper than 3 nm and with a diameter of about 15 nm. The density of the "pores" was approximately 20/ microm2 or 10 000 pores per thick dendrite. We believe these to be putative ion channels based on indirect evidence.

Hydration-scanning tunneling microscopy as a reliable method for imaging biological specimens and hydrophilic insulators.

The recently discovered high lateral conductivity of molecularly thin adsorbed water films enables investigation of biological specimens, and even of surfaces of hydrophilic insulators by scanning tunneling microscopy (STM). Here we demonstrate the capabilities of this method, which we call hydration-STM (HSTM), with images of various specimens taken in humid atmosphere: We obtained images of a glass coverslip, collagen molecules, tobacco mosaic virus, lipid bilayers and cryosectioned bovine achilles tendon on mica. To elucidate the physical mechanism of this conduction phenomenon we recorded current-voltage curves on hydrated mica. This revealed a basically ohmic behavior of the I-V curves without a threshold voltage to activate the current transport and indicates that electrochemistry probably does not dominate the surface conductivity. We assume that the conduction mechanism is due to structuring of water at the surface.

Atomic force microscope measurements and manipulation of Langmuir-Blodgett films with modified tips.

A simple method for rendering atomic force microscope tips and cantilevers hydrophilic or hydrophobic through glow discharge in an appropriate gas atmosphere is introduced. Force curves at different humidities of these modified cantilevers were taken on freshly cleaved mica (hydrophilic surface) and on a monolayer of dipalmitoylphosphatidylethanolamine transferred onto mica (hydrophobic surface) to characterize the behavior of the cantilevers on hydrophilic and hydrophobic surfaces. Furthermore, Langmuir-Blodgett bilayers, with a dipalmitoylphosphatidylethanolamine bottom layer and a dipalmitoylphosphatidylcholine top layer, were imaged in the constant force mode in a multimode atomic force microscope in air under controlled humidity conditions. The friction and elasticity signal were recorded parallel to the topography. By varying the force exerted by the tip on the sample, different layers of the Langmuir-Blodgett system could be removed or flattened. Removal exposed underlying layers that exhibited a different friction and elasticity behavior. Furthermore, force scans with tips rendered hydrophobic were taken on the different layers of the sample to characterize the hydrophilic/hydrophobic nature of the layers. Only by combining the results obtained by the different methods can the structure of the lipid layer systems be identified.