Segmental flexibility of immunoglobulin G antibody molecules in solution: a new interpretation

Abstract

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.

Long-Lifetime Ru(II) Complexes as Labeling Reagents for Sulfhydryl Groups

“…We conclude that the probe is tightly bound to HSA and its anisotropy decay reflects the overall rotation of the protein. Similarly, for IgG the recovered rotational correlation time of 108 ns represents rotational motion of the F ab segments, which were previously shown to display correlation times near 18 and 100 ns (27). A single correlation time adequately described these data as determined from the residuals and R 2 .…”

The Relative Rate of Immunoglobulin Gamma 1 Fragmentation

“…[4][5][6][7][8][9] The human IgG1 hinge region consists of 17 residues and may be subdivided into upper, core, and lower hinge regions. 10 The upper hinge includes residues D 221 KTHT 225 (Eu numbering 11 ) and has been shown to be solvent exposed and highly flexible with little to no structure (disordered) for IgG1 b12.…”

Pyrene-Labeled DNA Probes for Homogeneous Detection of Complementary DNA Sequences: Poly(C) Model System

“…However, we assume that these The changes in I VV , I VH , I(54.7), A, and lifetimes equations apply to a first approximation to the case of which occur when pyrene-poly(C) hybridizes with po-multiple lifetimes if we use the average lifetime of Eq. ly(I) can be related to each other through the following [3] in place of t in these equations (12). Furthermore, steady-state relationships which apply strictly to the we assume that the equations can be used for practical case where the fluorophore displays a single lifetime t, estimates in cases of restricted rotational motion where is free to rotate in all direction (unrestricted rotational the restriction is not severe (low fluorescence anisotmotion) (6,11), and is not subject to static quenching.…”

“…This Equations [4] and [5] and [7] to [12] show that the effects of t and anisotropy A on the steady-state fluo-pyrene-labeled poly(C) displays smaller changes in intensity upon hybridization than the sulfonylchloriderescence intensities I VV and I VH can be separated or factored as individual terms. The lifetime factor is the labeled poly(C).…”