THE STRUCTURE DIFFERENCE OF PROTEINS ISOLATED ON SUBSTRATE WITH DIFFERENT TECHNIQUES AS STUDIED BY THE ATOMIC-FORCE MICROSCOPE

The height and width of proteins deposited on mica substrates were mea sured from cross sections of their atomic force microscope (AFM) image s. The tapping mode AFM gave very stable and reproducible images for h igh molecular weight proteins. The following results were obtained: (1 ) The thickness of mono-, bi-, and trilayered purple membranes was 5.3 , 10.4, and 16.0 nm, respectively. The calibration curve of z range of AFM based on the above data was linear. The deviation of the calibrat ion curve at the origin was < 0.6 nm. (2) The height of slow form alph a-2-macroglobulin (alpha 2M) molecule changed depending on sampling me thods. When the protein was freeze-dried on a mica substrate prewetted with water, alpha 2M gave the highest value for its height. The fact that freeze-drying, especially after prewetting of the substrate, was effective to prevent flattening of the molecule suggested that sample deposition must be as gentle as possible to keep the original height o f the molecules. (3) Furthermore, we compared differences of height an d width between alpha 2M and myosin filament. The result suggested tha t alpha 2M had a disk-like rather than a spherical form. Large protein s such as alpha 2M are still difficult to crystallize for x-ray analys is, and we tested the AFM method for the study of minute height differ ences of such proteins.