Segmental flexibility of immunoglobulin G antibody molecules in solution: a new interpretation.
We propose a new model for the segmental flexibility of immunoglobulin G (IgG). The flexibility of native and mildly reduced anti-5-(dimethylamino)naphthalene-1-sulfonyl (anti-dansyl) antibody was reexamined by nanosecond fluorescence spectroscopy using deconvolution and lamp-shift corrections. The rabbit antibodies used for this study were purified of dimers and other aggregates. The original results indicated that the decay of fluorescence anisotropy involved two rotational correlation times. It was suggested that the short rotational correlation time, phi s, represented a flexible Fab arm motion over a restricted angle and that the long correlation time, phi L, represented global tumbling of the molecule [Yguerabide, J., Epstein, H. F., & Stryer, L. (1970) J. Mol. Biol. 51, 573--590]. Our new data indicate that the long correlation time primarily represents motions of the Fab segments and not global tumbling of the entire molecule. This interpretation implies a more flexible model for IgG. Thus, in solution the antibody arms appear to move over a wide angle and are not restricted to 33 degrees as was suggested in the earlier model. Simple diffusion calculations and other evidence suggest that phi s may represent V-module flexibility about the switch peptides or Fab twisting around its long axis, whereas phi L may represent wagging or wobbling motions of the Fab arms about the hinge region. The faster motions appear to occur over small angles whereas the slower wagging or wobbling motions responsible for most of the decay of anisotropy appear to be much less restricted. The biological function of IgG and anisotropy changes resulting from hinge disulfide cleavage are interpreted in terms of the proposed model. We also demonstrate a useful method for comparison of time-dependent and steady-state fluorescence polarization data.
D000818:Animals; D003619:Dansyl Compounds; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D008433:Mathematics; D011487:Protein Conformation; D011817:Rabbits; D012710:Serum Albumin, Bovine; D012996:Solutions; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D012996:Solutions; D012710:Serum Albumin, Bovine