STRUCTURAL AND DYNAMIC PROPERTIES OF THE FV FRAGMENT AND THE SINGLE-CHAIN FV FRAGMENT OF AN ANTIBODY IN SOLUTION INVESTIGATED BY HETERONUCLEAR 3-DIMENSIONAL NMR-SPECTROSCOPY

F-v fragments, heterodimers of the variable light (V-L) and variable h eavy chain (V-H) domains, are the smallest functional antibody units w ith molecular masses of similar to 26 kDa. The structural and dynamic properties of the F-v fragment and the corresponding single-chain F-v fragment (scF: V-H-linker-V-L, 252 amino acids) of the phosphorylcholi ne-binding antibody McPC603 in the presence of hapten have been studie d in solution by heteronuclear multidimensional NMR spectroscopy. Both N-15 TOCSY-HMQC and triple-resonance experiments (HNCA and HN(CA)H, w ith N-15-C-13-labeled protein) gave poor spectra, due to short T-2 rel axation times for most of the backbone H-1, N-15, and C-13(alpha) atom s. The assignment procedure therefore relied upon the combination of a mino acid and domain (V-L) specifically labeled spectra and the 3D NOE SY-HMQC spectrum of the uniformly N-15 labeled F-v and scF(v) fragment s. Approximately 80% of the N-15 and H-1 backbone and 60% of the H-1 s ide-chain resonances have been assigned. Short- and long-range NOEs we re used to determine the extent of beta-sheet structure and were compa red to the X-ray crystallographic data. The H-1-N-15 NOE data indicate that the scF(v) backbone has a well-defined structure of limited conf ormational flexibility. However, the linker of the scF(v) fragment exh ibits substantial fast internal motion (on the picosecond to nanosecon d time scale) compared with the overall rotational correlation time of the whole molecule. Several residues in the CDRs, in turns, or at the C-terminal end of the protein have smaller NOEs, reflecting some degr ee of rapid motion in the protein backbone.