Introduction 1 2 3 4 5 4 6,7 8–11 12–16 3 2 17,18 19 17,18,20 12,16 21–23` 24–26 26 Fig. 1 Supplementary Fig. 1 26 26 27 − 1 Results Thermodynamic stability Wild-type FNoTNc G D–N − 1 − 1 G D–N − 1 17 Anomalous response of certain peripheral mutations Fig. 2 G D–N Fig. 2 Fig.
                            2 G D–N Supplementary Table
                        1 Equilibrium hydrogen exchange k ex − 2 − 4 − 1 k ex G ex app Supplementary Table
                            2 G ex app Fig.
                                3 Folding kinetics Wild-type FNoTNc Fig. 4 Fig. 4 − 1 m − 1 − 1 G D–N − 1 m − 1 − 1 m T Φ-value analysis 28–30 Supplementary Fig.
                            2 G D–N Table
                                1 Dynamics Backbone dynamics 15 T 1 T 2 1 15 1 15 Supplementary Table 3 S 2 R ex Fig.
                                5 Supplementary Table 4 S 2 R ex Side-chain dynamics I z C z D z I z C z D y I z C z T 1 T 2 31 Supplementary Table
                            5 S 2 22 Supplementary Table
                            6 S 2 S 2 S 2 Fig. 6 Discussion The core of FNoTNc is similar to that of TNfn3, but apparently less closely
                    packed. Evidence from mutations Fig. 7 3 versus 3 G D–N Fig. 7 32,33 G D–N Evidence from side-chain dynamics 22,23 Fig. 6 Behaviour of peripheral regions of the protein is modulated by
                        the surface and loops Evidence from mutation G D–N 19 G D–N Fig. 2 Fig. 2 Evidence from hydrogen exchange 19 34,35 Fig. 3 The stability of FNoTNc is modulated by both the core and the
                        surface Fig.
                        7 36 et
                            al. 37 The TNfn3 core governs the folding kinetics Wild-type kinetics Fig. 4 8,38 8,39 18 30,40,41 42 k f 2 − 1 k u 2 − 4 − 4 − 1 − 5 − 1 Φ-value analysis 43 12 16 Table 1 12 16 Fig. 8 Dynamics from NMR is determined by local interactions Side-chain dynamics The side-chain dynamics appear to reflect core packing, as was
                            discussed above, and so FNoTNc resembles TNfn3 more closely than
                        FNfn10. Backbone dynamics S 2 Fig. 5 37 S 2 et al. 37 S 2 S 2 Conclusion We have grafted the core of one fnIII domain (TNfn3) into the homologue
                    FNfn10, creating a chimera, FNoTNc, which has retained the structure of the
                    parent proteins. Using several different probes, we have shown that FNoTNc does
                    not behave like either one of the parent proteins alone. Instead, it has
                    retained a number of properties of each. We find that each property investigated
                    clearly resembles the behaviour of one of the parents, enabling us to separate
                    the contribution of the core and the surface of the protein in determining the
                    behaviour of the domain. Some of these are unsurprising, such as the pH
                    dependence of stability, the core side-chain dynamics and the dependence of
                    folding on the composition of the core. However, the surface of the protein
                    confers significant stability not only on the native state, but also on the
                    transition state for folding. Others properties are less predictable. The
                    surface of the protein confers “plasticity” in peripheral regions of the
                    proteins as detected by the anomalous response of some regions of the core to
                    mutation and hydrogen exchange protection patterns. This suggests that the surface of a domain may have a more significant
                    coupling with the core than we had previously considered. Since most biophysical
                    studies tend to focus on the core of a protein, this coupling is a relatively
                    unexplored area of research. Materials and Methods Chemicals GdmCl was purchased from MP Biomedicals Inc., guanidine isothiocyanate
                        from Gibco-BRL and urea from BDH Laboratory Supplies. Protein expression and purification Table 1 Supplementary Table 1 20 Measurements of protein stability 20 Kinetic measurements 12 Φ-value analysis (1) 43 (1) Φ = Δ Δ G D – TS Δ Δ G D – N Δ Δ G D – TS = R T ln ( k f WT k f mut ) k f WT k f mut NMR sample preparation 17 15 13 15 15 4 13 22 Chemical shift assignments 13 15 2 1 15 1 13 44,45 1 15 Supplementary Fig. 3 Supplementary Table 7 2 22,46,47 1 13 1 13 1 13 13 Supplementary Fig. 4 Supplementary Table 7 Hydrogen exchange 15 2 48 G ex app k ex k int 49,50 (2) † 51 (2) Δ G ex app = − R T ln k ex k int 15 15 T 1 T 2 1 15 22 2 T 1 T 1ρ I z C z I z C z D z I z C z D y 22,31 Appendix A Supplementary Data Supplementary Figures  Supplementary Tables  Appendix A Supplementary Data doi:10.1016/j.jmb.2007.10.056