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 dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel 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 calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level.
Schneider
S W
SW
Department of Physiology, University of Münster, Robert-Kochstrasse 27a, D-48149 Münster, Germany.
Lärmer
J
J
Henderson
R M
RM
Oberleithner
H
H
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D001792:Blood Platelets / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007075:Immunoglobulin M / Q000737:chemistry; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D018625:Microscopy, Atomic Force; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry; D024661:Potassium Channels, Inwardly Rectifying; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry; D000098662:Vasodilator-Stimulated Phosphoprotein
D001457:Anion Exchange Protein 1, Erythrocyte; D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; C086989:KCNJ1 protein, human; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D010750:Phosphoproteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D000098662:Vasodilator-Stimulated Phosphoprotein; D013049:Spectrin; D005982:Glutathione Transferase