MOLECULAR-WEIGHTS OF INDIVIDUAL PROTEINS CORRELATE WITH MOLECULAR VOLUMES MEASURED BY ATOMIC-FORCE MICROSCOPY

Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three d imensions. We have used AFM to measure the molecular volumes of a numb er of proteins and to determine any correlation with their known molec ular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium c hannel protein ROMK 1, the immunoglobulins IgG and IgM. and the vasodi lator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-g el electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculat ed volumes of the individual proteins. The degree of resolution achiev ed (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment o f the: proteins to the mica. This was aided by coating the mica with s uitable detergent and by imaging using the AFM tapping mode which mini mizes any lateral force applied to the protein. We conclude that singl e (native and denatured) proteins can be imaged by AFM in three dimens ions and identified by their specific molecular volumes. This new appr oach permits detection of the number of monomers of a homomultimeric p rotein and study of single proteins under physiological conditions at the molecular level.