Protein dynamics and the immunological evolution of molecular recognition

Abstract

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) ...

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics

“…We also reproduced the results of previous 3PEPS studies (30) 2a). The presence of the static inhomogeneity demonstrates that the germ-line Ab populates a broad distribution of different combining-site conformations that do not interconvert on the time scale of the experiment (0.3 ns).…”

“…All Abs were expressed as Fab fragments (29,30,37). After isolation from the cell lysates by protein G affinity chromatography, Ab Fab fragments were further purified by cation ex- change chromatography (Mono S; Amersham Pharmacia, Piscataway, NJ).…”

“…Protein flexibility results from fluctuations between CSs that occur on the ps to ns time scale, and conformational heterogeneity results from transitions between different conformations that occur on longer time scales (25,26). One approach to experimentally characterizing protein flexibility is based on measuring how a protein relaxes after displacement from equilibrium by a photoinduced change in the charge distribution of a bound chromophore (27)(28)(29)(30). The induced motions are manifest as discrete peaks in the Ab spectral density, Ab ( ), which is the frequency domain representation of the ensembleaveraged time-correlation function of the electronic transition energy gap, M(t) (31).…”

“…5, which is published as supporting information on the PNAS web site). We prepared the chimeric Ab consisting of the germ-line light chain and mature heavy chain (V L gl V H 4-4-20 ) and used surface plasmon resonance and 3PEPS to demonstrate that the two V L mutations increase affinity and shift Ab ( ) to higher frequency (30). We now report production and characterization of the full germ-line Ab (V L gl V H gl ) and all of the heavy-chain single-point mutants.…”

“…This process resulted in a significant rigidification of the combining site, which increased the barrier to interconversion with other conformations. Finally, the two V L mutations, H L 34R and L L 46V, introduced and optimized an H bond between the protein and Fl (30). Thus, during evolution an appropriate combining site was first selected from an ensemble of conformations populated by the flexible germ-line receptor and then the selected combining site was further optimized for recognition of the target molecule.…”