Supplementary material Protein Expression and Purification En-HD L16A was expressed in a pRSET-derived pHisGro vector (Invitrogen). The En-HD gene within this vector was fused to the C-terminus of histidine-tagged GroEL apical domain connected via a TEV protease cleavage site. The fusion protein was then over-expressed at 37 oC as inclusion bodies in E. coli C41 cells using K-MOPS minimal media supplemented with 15NH4Cl (1g/L of culture) and 13C6-glucose (4g/L) as only nitrogen and carbon sources appropriately for samples. Expression in 100% 2 H2O M9 media with 15 N- or 12 15 N/13C-labelled C-2H7-glucose yielded perdeuteration of all nonlabile protons. Unlabelled protein was expressed in TTY media. After harvesting, the cells were collected by centrifugation, resuspended and sonicated. Cell debris, pelletted by centrifugation, was resuspended in 8 M urea. A mixture of resuspended debris and soluble lysate was loaded on a Ni-NTA column. The imidazole eluate was digested with TEV protease and purified using ionexchange chromatography followed by reverse-phase chromatography and lyophilisation. Resuspended protein was dialyzed against water, again lyophilised and dissolved in the appropriate buffer for use in the experiments. Residual Dipolar couplings (RDCs) of the denatured state We compared the RDCs of L16A with the crystal structure of wild-type Engrailed Homeodomain and the structure we determined of the denatured state of L16A. We used the method implemented in PALES 1 to find the best alignment tensor based on the whole of the analysed fragment or just the H2/H3 fragment. The correlation coefficient of 0.98 (Q factor = 0.19 2) between the determined L16A RDCs at 25oC and wild-type predicted RDCs for the H2/H3 part of the protein was similar to ones found for proteins with high-resolution crystal structures 3. This, by itself, suggests that the backbone structure of this motif did not change between the wild-type and the mutant. The agreement of L16A RDCs with the 10-50 fragment of the WT protein was much lower, which suggests that the average orientation of the N-terminal fragment must be different from that in the wild-type protein. The correlation between predicted and determined RDCs for H2/H3 of L16A is 0.85 (Q = 0. 46) for the acrylamide gels and 0.94 (Q = 0.33) for the Pf1 phages, which is still relatively high, but lower than similar correlation for the wild-type protein. We ascribe it to the inherently lower quality of NMR structures. The correlation for the whole protein is low, but there are insufficient long range NOEs to define the orientation of Helix 1 with precision. The RDCs for L16A are in Supplementary Figure 1. Dynamics of the denatured state The three values obtained, J(0), J(N) and J(0.87H) are indicators for overall as well as internal mobility on a timescale of ns to ps. J(0)eff values are, furthermore, affected by conformational exchange on a timescale lower than the overall tumbling time of the protein (ms-s). Overall inspection reveals little difference in J(0) between L16A and WT, implying similar overall tumbling times and compactness of L16A and WT. However, a general decrease of J(N) and an increase of J(0.87H) at 25oC indicate a shift of the frequency spectrum for L16A towards higher (ps) frequencies associated with a general increase in internal mobility. This is, in particular, apparent at the Nand C- termini where a more rapid decrease of J(0), J(N) spectral densities in L16A suggests substantial invasive fraying of the residual helices. References 1. Losonczi, J. A., Andrec, M., Fischer, M. W. & Prestegard, J. H. Order matrix analysis of residual dipolar couplings using singular value decomposition. J. Magn. Reson. 138, 334 - 342 (1999). 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Cornilescu, G., Marquardt, J. L., Ottiger, M. & A., B. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid Crystalline Phase. J. Amer. Chem. Soc. 120, 6836 - 6837 (1998). Bax, A. Weak alignment offers new NMR opportunities to study protein structure and dynamics. Protein Sci 12, 1-16 (2003). Tjandra, N., Szabo, A. & Bax, A. Protein backbone dynamics and N-15 chemical shift anisotropy from quantitative measurement of relaxation interference effects. J. Amer. Chem. Soc. 118, 6986-6991 (1996). Fushman, D., Tjandra, N. & Cowburn, D. Direct measurement of N-15 chemical shift anisotropy in solution. J. Amer. Chem. Soc. 120, 10947-10952 (1998). Mayor, U., Grossmann, J. G., Foster, N. W., Freund, S. M. & Fersht, A. R. The denatured state of Engrailed Homeodomain under denaturing and native conditions. J. Mol. Biol. 333, 977-991 (2003). Hansen, M. R., Mueller, L. & Pardi, A. Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat. Struct. Biol. 5, 1065 - 1074 (1998). Tycko. Alignment of biopolymers in strained gels: a new way to create detectable dipole-dipole couplings in high-resolution biomolecular NMR. J. Amer. Chem. Soc. (2000). Cordier, F., Dingley, A. J. & Grzesiek, S. A doublet-separated sensitivityenhanced HSQC for the determination of scalar and dipolar one-bond Jcouplings. J. Biomol. NMR 13, 175 - 180 (1999). Goddard, T. D. & Kneller, D. G. Sparky 3, University of California at San Francisco. Zweckstetter M. & Bax, A. Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Amer. Chem. Soc. 122, 3791 - 3792 (2000). Supplementary Table 1. Summary of conformational restraints and statistics for the 25 accepted structures of En-HD L16A. A. Structural constraints Sequential NOEs (1≤|i – j|<2) 355 Medium range NOEs (2≤|i – j|<5) 415 Long range NOEs (|i – j|≥5) 91 Total NOEs 861 B. Statistics for the accepted structures E (overall) 185 ± 8 E (bonds) 8±1 E (angles) 59 ± 6 E(impropers) 8±2 E (van der Waals) 73 ± 4 E (noes) 36 ± 5 Average atomic backbone RMSD (Å) from the mean structure Residues 28 – 53 0.87 ± 0.33 Residues 31-51 0.70 ± 0.28 Residues 10 – 53 2.35 ± 0.83 Ramachandran analysis Residues in most favoured regions (%) 61.4 Residues in additional allowed regions (%) 22.1 Residues in generously allowed regions (%) 7.0 Residues in disallowed regions (%) 3.5 Supplementary Table 2. Comparison of EnHD L16A RDCs with the structure of wildtype and L16A. H1/H2/H3 H2/H3 (residues 10 – 50) (residues 31 – 50) 9% acrylamide 0.45 (0.73) 0.98 (0.19) 12.5% Pf1 0.79 (0.61) 0.98 (0.18) WT: 'Best fit' WT: Using alignment tensor predicted from H2/H3 9% acrylamide 0.38 (0.91) 0.98 (0.19) 12.5% Pf1 0.77 (0.85) 0.98 (0.18) 9% acrylamide 0.71 (0.60) 0.85 (0.46) 12.5% Pf1 0.79 (0.58) 0.94 (0.33) L16A: 'Best fit' L16A: Using alignment tensor predicted from H2/H3 9% acrylamide 0.09 (1.1) 0.85 (0.46) 12.5% Pf1 0.77 (0.64) 0.94 (0.33) The correlation between RDCs determined for L16A and predicted for the WT is given the main number. The Cornilescu2 Q factor is given in brackets. High resolution crystal structures, like the one of EnHD WT, should give Q factor <0.25 if it represents well the bond vector orientations 3. The HN-N RDCs were measured in 12.5% Pf1 phage (at pH 7.0, 50mM KPi, 100mM NaCl) 7 and in 9% acrylamide gels (37:1 bisacrylamide:acrylamide ratio) 8 using DSSE method at 25oC9. The peak centres were picked using peak integration routine in Sparky 10. Comparison of the predicted and determined RDCs was done with PALES 11. Supplementary Figure Legend Supplementary Figure 1. HN-N RDCs for L16A at 298 K (calculated as D – J coupling). A: 9% acrylamide. B: 12.5% Pf1 phage at pH 7.0.