Electron microscopic location of protein thiol residues

Abstract

ATTEMPTS to use conventional transmission electron microscopy to visualise single heavy atoms attached to protein thiol residues are usually unsuccessful, because the signal due to the heavy atom is generally too weak to be detected against background noise and the signal due to the protein and negative stain (if used). This is unfortunate because such a technique would prove invaluable as a means of determining molecular orientations in multiprotein assemblies and relative positions of thiol residues within individual molecules. As one approach to this problem we have devised and report here a method by which the signal at the thiol residues can be increased by introducing several heavy atoms at each site. This considerably aids ultrastructural localisation by electron microscopy, particularly in the presence of heavy metal negative stains such as uranyl acetate. We illustrate the technique by visualising the thiol residues in uranyl acetate-negatively stained magnesium paracrystals of rabbit skeletal muscle tropomyosin. Although the conditions we report gave satisfactory results with tropomyosin, they should only be considered as a guide when labelling other proteins, as the optimum reaction conditions may be different in some cases.