Scanning probe microscopy (SPM) allows the visualization of single protein molecules and their complexes with membranes. In some cases even visualization of their inner details becomes possible. High spatial resolution achieved in SPM is not the only advantage of the new method. Even more important is the possibility to study proteins in various environments: air with controlled humidity, water and physiologically relevant buffers, organic solvents. External parameters - temperature, humidity, pressure, salt concentration - can be varied during measurements as well. This gives a unique opportunity to study conformational changes in proteins in situ [1].
 
 

Liposomes

Visualization of liposomes, proteoliposomes and vesicles 10 - 1000 nm in diameter makes it possible to analyze the size distribution. Processes of liposomes aggregation and fusion can be studied using SPM [2].
 
 
 

Fig. 1. Liposomes from phosphatidylcholin on graphite surface. Image provided by atomic force microscope. The size of individual liposome - 30 nm.

 

Antibodies

Immobilization of antibodies on solid supports is a primer task for creation of imunnosensors. Scanning probe microscopy (SPM) proved to be a useful tool for technological control during the preparation of specifically active films of biomaterials. Films of hepatitis B antibodies with amphyphylic polyelectrolytes created in the Center of Molecular Diagnostics and Treatment (Lab. of Dr. I.N. Kurochkin) sensible to HBs antigen were successfully visualized and quantitatively analyzed [3].
 

Fig. 2. Hepatitis B antibodies immobilized on graphite substrate using polyelectrolyte film. Image size 3.5x3.5 mm2.

 
 
 

Self-assembly in protein-membrane systems

Lipids, proteins and other biologically important substances form liquid crystals and quasicrystalline nanostructures. Visualization of such formations under various physiological conditions is of great help in the studies of lipid-protein and protein-protein concurrent interactions [4]. Self-assembled nanostructures form from membrane proteins, e.g. ATP-syntase and cytochrome P450 [5].
 
 
 

Fig. 3. Quasicrystalline formations from ATP-syntase membrane protein complex: nanotubes (image size 2.5x2.5 m m2).

 

Fig. 4. In liquid crystalline so-called "ripple" phase molecules of lipid are arranged in cylinders with parallel orientation (bands on the image). Individual protein molecules (cytochrome P-450 scc) are clearly distinguishable as well.

 
 
 

Scanning tunneling microscopy

Scanning tunneling microscopy (STM) makes it real to measure conductivity of biological specimen. It opens perspectives of registration of charge transfer on single molecule level.
 
 

Representative publications:

1. Kiselyova O.I., Yaminsky I.V. "Proteins and memrane-protein complexes" in Scanning Probe Microscopy of Biopolymers (Ed. by Yaminsky I.V., Scientific World, Moscow, 1997) p. 41. (in Russian).
2. Uvarov V.Yu., Ivanov Yu.D., Romanov A.N., Gallyamov M.O., Kiselyova O.I., Yaminsky I.V. Scanning tunneling microscopy study of cytochrome P450 2B4 incorporated in proteoliposomes // Biochimie 78 (1996) 780-784.
3. Budashov I.A., Kurochkin I.N., Tsibezov V.V., Kalnov S.L., Denisov A.K., Kiselyova O.I., Yaminsky I.V. Structural and Functional Properties of Langmuir Films from Antibodies based on Amphiphilic Polyelectrolytes // in press
4. Kiselyova O.I., Yaguzhinsky L.S., Yaminsky I.V., Yanushin M.F., Bueldt G. Quasicrystalline nanostructures formed on HOPG surface from ATP-syntase protein complex of thermophylic bacteria Chlorophlexus aurantiacus // Surface Science in press
5. Kiselyova O.I., Guryev O.L., Krivosheev A.V., Usanov S.A., Yaminsky I.V. Atomic force microscopy studies of Langmuir-Blodgett films of cytochrome P450 scc (CYP11A1): hemeprotein aggregation states and interaction with lipids // Langmuir in press