Line-shape analysis of NMR difference spectra of an anti-spin-label antibody.
Specifically deuteriated Fab fragments of the anti-spin-label antibody AN02 were prepared. NMR difference spectra were obtained, in which the spectrum of Fab with some fraction of the binding sites occupied with spin-label hapten was subtracted from the spectrum of Fab with no spin-label. The peak heights were analyzed as a function of the fractional occupation of the binding site, using a computer program that calculates a best fit to the observed spectra. This method treats all of the peaks in the spectra simultaneously. Analyzing all peaks at once allows for the interdependencies in the spectra arising from overlap of positive and negative signals from different peaks. The fitting program calculates line widths for the peaks arising from protons in the binding site region. Almost all of the line widths calculated for the spectrum of the Fab complex with diamagnetic hapten dinitrophenyldiglycine were found to be narrower than the line widths of the corresponding resonances in the spectrum of Fab with an empty binding site. The distances of the binding site region protons from the unpaired electron of the hapten were also obtained from this calculation. Two tyrosine protons were found to be close (less than A) to this electron. These line-width and distance results are discussed with respect to the structure and dynamics of the antibody binding site.
Frey
T
T
Stauffer Laboratory for Physical Chemistry, Stanford University, California 94305.
Anglister
J
J
McConnell
H M
HM
D007140:Immunoglobulin Fab Fragments; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D013113:Spin Labels / Q000276:immunology*
D007140:Immunoglobulin Fab Fragments; D013113:Spin Labels