NMR-detected conformational exchange observed in a computationally designed variant of protein Gβ1

Detailed biophysical characterization of computationally designed proteins has become increasingly important in order to thoroughly understand the properties of these variants compared with wild-type and to apply this knowledge to future designs. The protein dynamics and structural properties of a computationally designed variant (Δ1.5) of the β1 domain of streptococcal protein G (Gβ1) were measured using multinuclear NMR methods. Results from relaxation, diffusion and hydrogen exchange experiments indicate that the variant weakly self-associates at NMR concentrations, with evidence for multiple binding sites. Although comparison of fast (ps-ns) timescale motions shows only small differences in dynamics between Δ1.5 and wild-type, results from the measurement of intermediate (μs-ms) timescale motions are very different. Significant backbone conformational exchange has been observed in the variant at positions all along the sequence, whereas the wild-type Gβ1 shows little evidence for this type of motion. This increased conformational exchange in Δ1.5 has been attributed to core overpacking resulting from the incorporation of two large hydrophobic side chains and the loss of an aromatic T-stacking interaction. These data highlight, in detail, the potential consequences of incorporating major perturbations in the core of a protein and the need to carry out more detailed analyses of the biophysical properties of designed proteins in order to better understand and predict the effects of mutations.