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Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

Abstract

Many biochemical processes proceed through the formation of functionally significant intermediates1,2. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population1,2,3,4. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology1. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase5, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

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Figure 1: Measurement of kf and ku in the G48M and G48V Fyn SH3 domains.
Figure 2: Three-site folding model for G48M and G48V Fyn SH3.
Figure 3: Structural analysis of the I state of mutants G48M and G48V of Fyn SH3 using chemical shifts.

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Acknowledgements

This work was supported through grants from the Canadian Institutes of Health Research (A.R.D, L.E.K and D.M.K.), the European Comission (X.S.), the Royal Society (M.V.), the Leverhulme Trust (M.V. and C.M.D.) and the Wellcome Trust (C.M.D.). L.E.K. holds a Canada Research Chair in Biochemistry.

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Correspondence to Christopher M. Dobson or Lewis E. Kay.

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Supplementary information

Supplementary Information

Includes information on fitting of dispersion profiles and structure calculations; Table 1S. Folding kinetics parameters obtained from a global fit of CPMG dispersion data for Fyn SH3 mutants; Table 2S. X2 target functions obtained in global fits of CPMG dispersion data for 23(26) residues of G48M(G48V) measured at 5(4) temperatures and 3(2) magnetic fields using 2- and 3-site exchange models; Table 3S. Thermodynamic parameters for G48M(G48V) mutants of the Fyn SH3 domain obtained from a global fit of CPMG dispersion data using 3- and 2-site exchange models. Table 4S. Backbone 15N chemical shift differences between states F and I (δFI) and between states F and U (δFU) and their ratio Δexp=δFI/δFU for G48M and G48V mutants of the Fyn SH3 domain; Figure 1S. Average r.m.s deviation from the native state as a function of residue number for the calculated I states of G48M and G48V; Figure 2S. Relaxation dispersion curves recorded for G48M at concentrations of 1.0 and 0.3 mM, 15 and 30oC, do not depend on protein concentration. (PDF 282 kb)

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Korzhnev, D., Salvatella, X., Vendruscolo, M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004). https://doi.org/10.1038/nature02655

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