While it is accepted that protein flexibility plays a role in protein folding, catalysis, and molecular recognition, few techniques are capable of the rigorous measurement of protein motions required to quantify flexibility. Three-pulse photon echo shift spectroscopy can be used to measure the time scale of protein motions, and we have used this technique, along with steady-state spectroscopy and binding and structural data, to examine the immunological evolution of protein flexibility in an anti-fluorescein antibody. Two light chain somatic mutations increase affinity for fluorescein by 12-fold but also significantly affect flexibility. Specifically, a rigidification of the protein is seen in each of three observable motions; two slower motions undergo decreased amplitudes of displacement, by 3-and 20-fold, respectively, in response to an applied force, and the distribution associated with the amplitude of a faster motion is narrowed upon somatic mutation. The somatic mutations appear to rigidify the antibody-fluorescein complex by more strongly anchoring fluorescein to the protein and by more tightly packing the complex. The data demonstrate that in addition to affinity, antibody dynamics are systematically manipulated during affinity maturation, and they imply that the evolution of protein flexibility may be a central component of the immune response. The results also reflect the type of protein rigidification that may be important for other biological interactions, such as protein-protein, protein-ligand or protein-drug, and enzymesubstrate recognition. M odels of molecular recognition, based on conformational selection (1-3), induced-fit (4-6), or lock-and-key-type mechanisms (5, 7), are central to describing virtually all proteinprotein and protein-ligand interactions. However, these models have been difficult to test, because they differ only in the flexibility of the protein, and protein flexibility has been difficult to quantify (8). Thus, studies of molecular recognition have not focused directly on flexibility, but instead have searched for manifestations of it, such as changes in on or off rates, varying binding entropies, and structural rearrangements. Nowhere is molecular recognition more important than in the immune system, where a finite number of receptors [antibodies (Ab) and T cell receptors] must bind a virtually infinite range of foreign molecules and peptides (9-16).While it is apparent that during the initial stages of an immune response, there must be Ab present that have a broad range of specificities, Ab may also be isolated that are highly specific. These highly specific Ab are typically isolated in the later stages of an immune response and are usually highly mutated compared with their corresponding germ-line gene sequences (17). It has thus been argued that during affinity maturation, somatic mutations act to rigidify the Ab, leading to more specific epitope recognition (9,13,(18)(19)(20)(21)(22)(23)(24). In this model, the binding sites of germ-line Ab (Ab before somatic mutation) ...