The substructure of immunoglobulin G resolved to 25 kDa using amplitude modulation AFM in air.
Amplitude modulation (or tapping-mode) atomic force microscopy (AM AFM or TM AFM) in air can reveal sub-molecular details of isolated multi-subunit proteins, such as immunoglobulin G (IgG) antibodies, on atomically flat support surfaces such as mica [A. San Paulo, R. Garcia, Biophys. J. 78(3) (2000) 1599]. This is achieved by controlling the microscope imaging parameters (e.g. cantilever drive frequency and set-point amplitude) to keep the AFM tip predominantly in the attractive force regime. Under these conditions, the 50 kDa F(c) and F(ab) subunits can be resolved when the molecule has the appropriate orientation on the surface. The presence of a water layer on hydrophilic mica is an important factor affecting imaging contrast, a consequence of capillary neck formation between tip and surface [L. Zitzler, S. Herminghaus, F. Mugele, Phys. Rev. B 66(15) (2002) 155436]. Desiccation of samples to remove surface bound water layers can yield reproducible imaging of the IgG substructure [N.H. Thomson, J. Microsc. (Oxford) 217(3) (2004) 193]. This approach has also given higher resolution than previously achieved, down to about 25 kDa, and these data are detailed here. These subdomains are formed as two immunoglobulin folds from the light and heavy peptide chains of the IgG crossover. This result has been validated by comparing the AFM images with X-ray crystallography data from the protein data bank. These data show that the AFM can obtain 25 kDa resolution on isolated protein molecules with commercially available silicon tips, but, as expected for a local probe technique, resolution is highly dependent on the macromolecular orientation on the support surface.
Thomson
Neil H
NH
Institute of Molecular Biophysics, School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK. N.H.Thompson@leeds.ac.uk
D000388:Air; D000818:Animals; D027081:DNA Gyrase / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force; D017434:Protein Structure, Tertiary
D007074:Immunoglobulin G; D027081:DNA Gyrase