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Published September 2008 | Supplemental Material
Journal Article Open

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.

Additional Information

© 2008 Oxford University Press. PEDS Advance Access originally published online on June 26, 2008. Received May 21, 2008; revised May 21, 2008; accepted May 23, 2008 Edited by Frances H. Arnold The authors would like to thank Drs Julie Forman-Kay, John Love and Kevin Plaxco for insightful comments on the manuscript, as well as Drs Andrew Chong, Kevin Gardner, Lewis Kay, Dorothee Kern, Gregory Lee and Leo Spyracopoulos for advice on experiments and data analysis. We are also grateful to Dr Scott Ross for NMR assistance and Mr. Ben Allen for his computational expertise. Funding Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship to K.A.C.); Howard Hughes Medical Institute. Supplementary data, including R1, R2° (transverse relaxation rate constant that is free of chemical/conformational exchange contributions), NOE, {eta}xy, {eta}z and Rex data are provided for wild-type protein Gβ1 and for the {Delta}1.5 variant at two concentrations, and are available at PEDS online.

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