th  - 1988 - 51st ENC Conference

I THU 9:40 I
NMR STUDIES OF ANTI-SPIN LABEL MONOCLONAL ANTIBODIES: D. J. Leahy, G. S. Rule',
and H. M. McConnell, Dept. of Chemistry, Stanford University, Stanford, CA, 94305.
Current progress towards the application of NMR to the study of proteins has been largely
restricted to protein molecules of low (10-15 KDa) molecular weight. We are currently utilizing
a number of approaches to obtain interpretable proton NMR spectra from a large protein, the Fab
fragment of an immunoglobulin.
The NMR spectra are simplified by methods of deuteration. We deuterate the Fab fragment
With both perdeuterated and partially deuterated amino acids. The partial deuteration results in the
removal of J coupling between protons. Thus we can observe and study individual resonance lines
from aromatic protons of Tyr and Trp residues, and the methyl groups of Val, Leu, lie, Thr, or Ala
residues.
The proton NMR spectra are further simplified by utilizing a spin-labelled hapten to
selectively broaden NMR signals from protons near the combining site. Chemical exchange of the
hapten modulates this broadening effect and spectra obtained at different occupancy levels can yield
measurements of the distance between the hapten and the amino acid residues in the range of 8-17
A.
By combining distance measurements, resonance assignments, and molecular modelling we
intend to develop a working model of the antibody combining site.
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