Motions of Immunoglobulins

In order to study IgE dynamics, the antigen dan l-lysine was bound to the antigen binding sites on the Fat, regions. Tlie 

Domain motions within the IgE molecule are evident from the multiexponential fits to the anisotropy decays (Table IM). When free in solution, IgE displays two correlation times of 48 and 125 ns. The larger value is due to overall rotation of IgE, and the shorter value is due to independent motions of the Fa, fragments. Assignment of the 48-ns correlation time to Fab motion is supported by the same correlation time being present when the antibody is 

Anisotropy decays of intrinsic and extrinsic probes frequently show subnanosecond components, which are usually due to rapid segmental motions. However, such components should be interpreted with caution and can be due to scattered light reaching thedetector. Another origin of rapid anisotropy components is the presence of unbound probe in a sample thought to contain only the labeled macromolecule. A free probe will typically display a 50-100-ps Correlation time, which can easily be mistakenly attributed to segmental motion. 

The effect of free probe is illusfrated by anisotropy decaysoftheyellowfluorescentproteinfYFP)from Vibrio fischeri. Hiis protein is from a bioluminescent bacterium, and the emitting fluon hore is FMN. The intensity decay time of FMN is 4.4 ns in solution and 7.6 ns when bound to YFP. The binding constant of FMN to YFP is only modest, so diat, depending on YFP concentration, scxne of the FMN can dissociate. 

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Anisotropy measurements are generally useful for measuring any process which increases or decreases the rate or extent of rotational diffusion. These processes include domain motions of immunoglobulins [21], denaturation of proteins [22] and the association of proteins with membranes [10]. Additionally, there are numerous applications of anisotropy measurements to membranes, in which the phase state and apparent fluidity are estimated from the anisotropy of probes which are bound to the membranes [23,24]. 

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