CHARACTERIZATION OF THE BACKBONE DYNAMICS OF AN ANTIDIGOXIN ANTIBODY-VL DOMAIN BY INVERSE DETECTED H-1-N-15 NMR - COMPARISONS WITH X-RAY DATA FOR THE FAB

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V(L) domain has been characterized by measurements of N-15 T1 and T2 relaxation times, H-1-N-15 NOE values, and H-1-H-2 exchange rates. These data were acquired with 2D inverse detected heteronuclear H-1-N-15 NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond time-scale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond time-scale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured H-1-H-2 exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of beta-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V(L) domain, including the three hypervariable loops, undergo dynamic changes upon V(L):V(H) association and/or complexation with digoxin. Overall, the 26-10 V(L) domain exhibits relatively low flexibility on the ps-ns timescale. The possible functional consequences of this result are considered.