Probing specific molecular conformations with the scanning force microscope. Complexes of plasmid DNA and anti-Z-DNA antibodies.
An anti-Z-DNA IgG antibody was used to probe for the left-handed Z-DNA conformation of a d(CG)11 insert in a negatively supercoiled plasmid DNA (pAN022). The complexes were spread on mica in the presence of a quaternary ammonium detergent benzyldimethylalkylammonium chloride and imaged with a scanning force microscope (SFM). The high affinity anti-Z-DNA antibody was retained even after restriction endonuclease cleavage of the DNA. The two arms in the product molecules had unequal lengths in conformity with the known location of the Z-DNA forming insert. Most complexes exhibited one IgG per DNA molecule. The bound antibodies were up to approximately 35 nm in diameter and extended approximately 2 nm from the mica surface. They were generally in a lateral orientation relative to the DNA, in accordance with prior chemical modification experimental data indicating a bipedal mode of binding for an anti-Z-DNA IgG. However, the SFM images also suggest that the DNA bends to accommodate the two Fab combining regions of the antibody. This study demonstrates the utility of the SFM for investigating conformation-dependent molecular recognition.
Pietrasanta
L I
LI
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Schaper
A
A
Jovin
T M
TM
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D004247:DNA / Q000737:chemistry* / Q000276:immunology; D004274:DNA, Recombinant / Q000737:chemistry / Q000276:immunology; D004278:DNA, Superhelical / Q000737:chemistry / Q000276:immunology; D004587:Electrophoresis, Agar Gel; D007091:Image Processing, Computer-Assisted; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism*; D008853:Microscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D010957:Plasmids
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D004278:DNA, Superhelical; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D004247:DNA