Protein dynamics and the diversity of an antibody response

Abstract

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.

FIGURE 1.

Structure of MPTS.

FIGURE 2.

Fit to steady-state absorption spectrum and 3PEPS decay of Ab 2E8 using the nonlinear response function formalism (circles, data points; lines, best fit to data; dotted lines, residuals).

FIGURE 3.

Anti-MPTS Ab sequences. The CDRs that form the Ab binding site are underlined. Kabat numbering is shown at the top (60).

FIGURE 4.

Evolutionary relationships between germ line (top) and affinity-matured (bottom) anti-MPTS Abs. For each Ab, the fractional amplitudes of elastic (λ_BO) and inelastic fluctuations (λ_K1,K2 and λ_inh) are shown in the pie chart and associated expansion, respectively. The bar diagram at the bottom shows the values of the time constant associated with barrier crossing between conformational substates (τ_K2) for each Ab (normalized to 13 ps). See “Discussion” and Fig. 9 for definitions of the parameters.

FIGURE 5.

Absorption spectra of Ab-MPTS complexes.

FIGURE 6.

3PEPS decays for the Ab-MPTS complexes. Lines are fits to the data.

FIGURE 7.

Spectral densities of Ab-MPTS complexes.

FIGURE 8.

Correlation between entropy and enthalpy of binding (T = 298 K).

FIGURE 9.

Schematic depiction of the protein fluctuations that give rise to λ_BO, ω_BO, Γ_BO, λ_K, τ_K, and λ_inh (41). SS 1–3 are different substates of conformations A and B.