Rotational dynamics of immunoglobulin G antibodies anchored in protein A soluble complexes.
The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, fS and fL, and the short rotational correlation time, phi S, were relatively small. On the other hand, the long rotational correlation time, phi L, increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG4-SpA2 complexes. Available electron microscopic data suggest that increases in phi L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in phi L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that phi L mainly represents flexible motions of the Fab segments and not global tumbling: the phi L-values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.
D000818:Animals; D000936:Antigen-Antibody Complex; D005454:Fluorescence Polarization; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011487:Protein Conformation; D011817:Rabbits; D012399:Rotation; D013050:Spectrometry, Fluorescence; D013205:Staphylococcal Protein A
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A