Table S1. Divalent metal ion and water coordinate distancesa,b

Ca2+-S100B Ca2+- D63NS100B TRTK-Ca2+-S100B TRTK-Ca2+- D63NS100B

Ca2+, pseudo EF HandSer-18 (C=O) 2.4 2.5 2.3 2.4Glu-21 (C=O) 2.4 2.4 2.3 2.4Asp-23 (C=O) 2.3 2.3 2.4 2.4Lys-26 (C=O) 2.4 2.5 2.4 2.4Glu-31 (Oε1, Oε2) 2.4, 2.6 2.5, 2.6 2.4, 2.7 2.4, 2.6H2O 2.3 2.4 2.3 2.3

Ca2+, canonical EF HandAsp-61 (Oδ1 or Oδ2) 2.3 2.2 2.2 2.3Asn/Asp-63 (Oδ1 or Oδ2) 2.3 2.4 2.4 2.3Asp-65 (Oδ1 or Oδ2) 2.4 2.5 2.4 2.4Glu-67 (C=O) 2.3 2.4 2.3 2.3Glu-72( Oε1, Oε2) 2.4, 2.6 2.5, 2.7 2.5, 2.7 2.4, 2.6H2O 2.4 2.5 2.4 2.4

Ca2+-bound H2OAsp-65 (Oδ1 or Oδ2)c 2.8 2.7 2.8 2.7Glu-67 (Oε1 or Oε2)d 2.7 2.4 2.5 2.6

a All distances are in Å and are from nitrogen, oxygen, or Ca2+.b Distances between the two Ca2+ ions for all four structures listed is 11.4 Å.c Distance from Asp65 side chain carboxylate oxygen to the water molecule at position 9 of the Ca2+ ion bound to EF2.d Distance from Glu67 side chain carboxylate oxygen to the water molecule at position 9 of the Ca2+ ion bound to EF1.

Table S2: TRTK-12-Ca2+-S100B 15N relaxation data, 14.1 TeslaPositio

nR1

600 R2600 NOE600

12 1.216 ± 0.061 13.819 ± 0.725 0.837 ± 0.0323 1.079 ± 0.064 14.437 ± 0.904 0.772 ± 0.0564 1.106 ± 0.051 14.082 ± 0.658 0.889 ± 0.0595 1.126 ± 0.045 14.162 ± 0.583 0.841 ± 0.0426 1.142 ± 0.024 14.246 ± 0.323 0.841 ± 0.0257 1.169 ± 0.039 15.228 ± 0.480 0.816 ± 0.0338 1.142 ± 0.029 13.946 ± 0.333 0.844 ± 0.0179 1.121 ± 0.021 14.248 ± 0.295 0.835 ± 0.026

10 1.143 ± 0.041 15.673 ± 0.616 0.763 ± 0.06211 1.099 ± 0.041 14.474 ± 0.571 0.868 ± 0.03512 1.145 ± 0.040 16.707 ± 0.689 0.865 ± 0.03113 1.078 ± 0.036 15.695 ± 0.596 0.832 ± 0.02914 1.385 ± 0.042 14.170 ± 0.240 0.878 ± 0.02415 1.084 ± 0.020 15.434 ± 0.620 0.850 ± 0.03116 1.171 ± 0.022 14.804 ± 0.300 0.818 ± 0.02117 1.114 ± 0.029 15.011 ± 0.436 0.820 ± 0.02218 1.114 ± 0.034 15.501 ± 0.522 0.886 ± 0.02919 1.421 ± 0.040 14.270 ± 0.423 0.852 ± 0.01920 1.202 ± 0.021 13.786 ± 0.247 0.855 ± 0.01721 1.118 ± 0.019 14.252 ± 0.262 0.855 ± 0.01722232425 1.441 ± 0.082 16.491 ± 1.066 0.885 ± 0.02926 1.185 ± 0.026 14.579 ± 0.350 0.842 ± 0.01827 1.148 ± 0.035 14.040 ± 0.457 0.872 ± 0.03028 1.178 ± 0.045 13.452 ± 0.503 0.848 ± 0.03129 1.524 ± 0.044 12.892 ± 2.395 0.852 ± 0.02730 1.663 ± 0.034 15.953 ± 0.160 0.874 ± 0.02031 1.164 ± 0.022 12.456 ± 0.218 0.832 ± 0.02032 1.201 ± 0.024 13.915 ± 0.294 0.873 ± 0.02233 0.880 ± 0.01834 1.232 ± 0.019 13.968 ± 0.217 0.839 ± 0.01935 1.231 ± 0.025 13.345 ± 0.281 0.838 ± 0.01936 1.165 ± 0.015 13.369 ± 0.165 0.807 ± 0.01737 1.224 ± 0.022 15.411 ± 0.286 0.814 ± 0.02638 1.201 ± 0.027 13.258 ± 0.315 0.847 ± 0.02339 1.171 ± 0.040 13.471 ± 0.510 0.874 ± 0.0314041 1.118 ± 0.031 14.149 ± 0.206 0.798 ± 0.03542 0.832 ± 0.06043 0.792 ± 0.05544 1.178 ± 0.02445 1.152 ± 0.043 13.512 ± 0.495 0.676 ± 0.05546 1.099 ± 0.015 11.472 ± 0.157 0.686 ± 0.02747 1.152 ± 0.032 11.682 ± 0.318 0.781 ± 0.03748 11.490 ± 2.594 0.730 ± 0.03149 1.093 ± 0.042 10.713 ± 0.388 0.505 ± 0.01450 0.823 ± 0.03951 1.196 ± 0.023 14.201 ± 0.269 0.779 ± 0.01452 1.213 ± 0.018 13.725 ± 0.231 0.815 ± 0.01953 1.075 ± 0.017 14.125 ± 0.251 0.777 ± 0.02454 0.886 ± 0.01755 1.138 ± 0.016 14.015 ± 0.214 0.806 ± 0.01756 1.138 ± 0.017 14.048 ± 0.218 0.831 ± 0.02057 0.839 ± 0.01758 1.098 ± 0.013 13.910 ± 0.174 0.828 ± 0.01659 1.124 ± 0.016 13.131 ± 0.195 0.823 ± 0.018

Table S3: TRTK-12-Ca2+-S100B 15N relaxation data, 18.8 TeslaPositio

nR1

800 R2800 NOE800

12 0.854 ± 0.041 21.313 ± 0.464 0.860 ± 0.0043 0.839 ± 0.083 21.537 ± 1.021 0.782 ± 0.0254 0.812 ± 0.055 21.889 ± 0.650 0.874 ± 0.0375 0.857 ± 0.057 20.561 ± 0.637 0.889 ± 0.0416 0.829 ± 0.038 18.236 ± 0.473 0.853 ± 0.0227 0.886 ± 0.0548 0.804 ± 0.033 20.631 ± 0.388 0.845 ± 0.0149 0.853 ± 0.043 18.736 ± 0.515 0.866 ± 0.00110 0.851 ± 0.046 19.804 ± 0.506 0.853 ± 0.00111 0.797 ± 0.096 20.878 ± 1.158 0.854 ± 0.00312 0.793 ± 0.132 23.297 ± 2.054 0.899 ± 0.03813 0.860 ± 0.110 22.007 ± 1.362 0.820 ± 0.01114 0.998 ± 0.070 18.535 ± 0.59115 0.874 ± 0.099 24.110 ± 1.288 0.888 ± 0.02016 0.907 ± 0.141 19.547 ± 1.579 0.847 ± 0.04017 0.868 ± 0.081 21.163 ± 0.974 0.857 ± 0.05818 0.795 ± 0.132 25.551 ± 2.714 0.874 ± 0.00719 1.012 ± 0.083 20.441 ± 0.969 0.845 ± 0.01320 0.865 ± 0.058 22.399 ± 0.677 0.869 ± 0.03021 0.857 ± 0.064 24.157 ± 0.844 0.836 ± 0.02322232425 1.111 ± 0.139 22.617 ± 1.529 0.881 ± 0.00426 0.883 ± 0.057 22.903 ± 0.686 0.853 ± 0.04027 0.858 ± 0.078 22.920 ± 1.003 0.856 ± 0.03828 0.900 ± 0.078 15.250 ± 0.852 0.866 ± 0.00129 1.019 ± 0.046 20.708 ± 0.723 0.871 ± 0.00230 1.116 ± 0.030 18.562 ± 0.305 0.813 ± 0.07231 0.852 ± 0.023 16.261 ± 0.239 0.860 ± 0.02332 0.906 ± 0.035 21.224 ± 0.395 0.838 ± 0.03333 0.992 ± 0.100 24.708 ± 1.154 0.847 ± 0.00534 0.872 ± 0.031 20.519 ± 0.371 0.874 ± 0.00135 0.857 ± 0.042 17.846 ± 0.480 0.873 ± 0.02936 0.921 ± 0.022 20.089 ± 0.254 0.872 ± 0.02137 0.917 ± 0.037 21.921 ± 0.456 0.860 ± 0.02938 0.908 ± 0.043 20.121 ± 0.464 0.874 ± 0.00339 0.858 ± 0.041 19.123 ± 0.435 0.760 ± 0.16740 0.905 ± 0.064 20.079 ± 0.521 0.839 ± 0.01241 0.887 ± 0.043 19.762 ± 0.463 0.865 ± 0.011

42 0.890 ± 0.071 22.979 ± 0.864 0.727 ± 0.02843 0.910 ± 0.053 18.477 ± 0.599 0.879 ± 0.00044 0.893 ± 0.073 25.199 ± 0.904 0.738 ± 0.02745 0.901 ± 0.069 18.418 ± 0.694 0.754 ± 0.00346 0.863 ± 0.030 15.598 ± 0.321 0.812 ± 0.03847 0.824 ± 0.054 22.035 ± 0.686 0.696 ± 0.01848 0.999 ± 0.045 17.269 ± 0.471 0.881 ± 0.00449 0.887 ± 0.013 15.824 ± 0.104 0.539 ± 0.01450 0.945 ± 0.060 25.566 ± 0.71951 0.891 ± 0.015 21.782 ± 0.192 0.804 ± 0.00752 0.848 ± 0.019 20.478 ± 0.245 0.806 ± 0.02453 0.816 ± 0.030 20.648 ± 0.347 0.823 ± 0.01654 0.843 ± 0.013 21.451 ± 0.126 0.808 ± 0.01955 0.797 ± 0.016 20.416 ± 0.183 0.839 ± 0.00156 0.785 ± 0.021 20.903 ± 0.256 0.804 ± 0.00757 0.801 ± 0.016 21.443 ± 0.19058 0.824 ± 0.015 21.696 ± 0.193 0.859 ± 0.02659 0.836 ± 0.017 19.706 ± 0.181 0.802 ± 0.01260 0.845 ± 0.024 21.396 ± 0.289 0.859 ± 0.02461 0.776 ± 0.021 20.953 ± 0.250 0.875 ± 0.01662 0.706 ± 0.027 16.573 ± 0.270 0.693 ± 0.00763 0.842 ± 0.014 18.592 ± 0.144 0.833 ± 0.00364 0.807 ± 0.015 18.352 ± 0.173 0.849 ± 0.00665 0.834 ± 0.119 25.363 ± 1.053 0.879 ± 0.00766 0.828 ± 0.019 19.170 ± 0.196 0.855 ± 0.00867 0.874 ± 0.014 21.147 ± 0.187 0.803 ± 0.01768 0.847 ± 0.062 15.455 ± 0.708 0.873 ± 0.01269 0.932 ± 0.074 21.063 ± 0.956 0.870 ± 0.02170 0.959 ± 0.081 23.040 ± 0.925 0.853 ± 0.01071 0.810 ± 0.038 19.841 ± 0.424 0.835 ± 0.01072 0.883 ± 0.046 18.169 ± 0.524 0.847 ± 0.00473 0.865 ± 0.043 19.292 ± 0.505 0.868 ± 0.01074 0.823 ± 0.050 21.735 ± 0.594 0.862 ± 0.02175 0.892 ± 0.052 22.757 ± 0.625 0.867 ± 0.01076 0.834 ± 0.034 19.884 ± 0.387 0.870 ± 0.00177 0.854 ± 0.034 21.215 ± 0.387 0.874 ± 0.00578 0.908 ± 0.061 22.563 ± 0.735 0.855 ± 0.01179 0.888 ± 0.041 16.660 ± 0.460 0.858 ± 0.00880 0.884 ± 0.036 21.232 ± 0.379 0.842 ± 0.00181 0.963 ± 0.095 21.950 ± 1.137 0.876 ± 0.02782 0.847 ± 0.01683 1.145 ± 0.022 15.967 ± 0.559 0.871 ± 0.00884 0.898 ± 0.00685 0.926 ± 0.0503 0.838 ± 0.00886 0.889 ± 0.107 18.812 ± 1.043 0.798 ± 0.01187 1.005 ± 0.1026 19.080 ± 0.987 0.777 ± 0.02088 0.974 ± 0.1522 16.460 ± 0.915 0.777 ± 0.00889 1.459 ± 0.0809 14.919 ± 0.470 0.489 ± 0.03190 1.498 ± 0.023 5.186 ± 0.115 0.316 ± 0.01791 0.926 ± 0.0503 18.812 ± 1.043 0.859 ± 0.026

Table S4: Ca2+-D63NS100B 15N relaxation data, 14.1 TeslaPositio

nR1

600 R2600 NOE600

12 1.343 ± 0.036 13.639 ± 0.369 0.785 ± 0.0583 1.177 ± 0.043 12.522 ± 0.504 0.819 ± 0.2004 1.239 ± 0.035 14.780 ± 0.455 0.780 ± 0.0005 1.195 ± 0.051 14.053 ± 0.664 0.748 ± 0.0046 1.157 ± 0.017 14.335 ± 0.201 0.809 ± 0.0457 1.285 ± 0.023 14.862 ± 0.3388 1.018 ± 0.021 13.931 ± 0.254 0.779 ± 0.0109 1.258 ± 0.039 15.815 ± 0.518 0.749 ± 0.003

10 1.179 ± 0.026 15.407 ± 0.363 0.837 ± 0.00011 1.301 ± 0.080 17.276 ± 1.112 0.799 ± 0.00112 1.158 ± 0.081 22.149 ± 1.902 0.895 ± 0.09913 1.123 ± 0.034 17.561 ± 0.575 0.815 ± 0.00414 1.392 ± 0.052 16.319 ± 0.659 0.827 ± 0.04215 1.165 ± 0.049 14.614 ± 0.682 0.844 ± 0.01716 1.234 ± 0.023 14.492 ± 0.294 0.786 ± 0.01317 1.175 ± 0.030 14.233 ± 0.363 0.809 ± 0.03418 1.088 ± 0.033 13.996 ± 0.454 0.876 ± 0.01219 1.340 ± 0.032 12.548 ± 0.305 0.844 ± 0.02220 1.226 ± 0.012 12.556 ± 0.132 0.801 ± 0.02221 1.111 ± 0.012 13.025 ± 0.149 0.770 ± 0.00422232425 1.590 ± 0.088 14.970 ± 0.960 0.820 ± 0.02226 1.199 ± 0.021 13.299 ± 0.247 0.830 ± 0.01927 1.196 ± 0.027 14.246 ± 0.340 0.823 ± 0.01928 1.297 ± 0.033 12.121 ± 0.302 0.850 ± 0.01029 0.827 ± 0.05630 0.824 ± 0.00631 1.215 ± 0.014 11.717 ± 0.123 0.793 ± 0.00832 1.309 ± 0.022 14.416 ± 0.221 0.852 ± 0.00433 1.255 ± 0.013 13.760 ± 0.137 0.831 ± 0.01834 1.341 ± 0.019 13.770 ± 0.188 0.841 ± 0.00935 1.256 ± 0.018 13.576 ± 0.189 0.834 ± 0.00536 1.292 ± 0.028 13.529 ± 0.234 0.782 ± 0.04837 1.306 ± 0.016 14.665 ± 0.197 0.804 ± 0.00938 1.280 ± 0.017 12.967 ± 0.179 0.828 ± 0.01939 1.239 ± 0.027 12.764 ± 0.255 0.834 ± 0.01840 1.041 ± 0.036 14.879 ± 0.589 0.861 ± 0.06241 1.163 ± 0.050 12.579 ± 0.774 0.813 ± 0.01242 ±43 1.396 ± 0.077 15.882 ± 0.88444 1.181 ± 0.063 16.896 ± 2.118 0.794 ± 0.13445 1.423 ± 0.164 7.925 ± 0.95146 1.256 ± 0.029 13.235 ± 0.313 0.607 ± 0.00947 1.455 ± 0.193 21.661 ± 3.81948 0.744 ± 0.05749 1.081 ± 0.031 9.658 ± 0.265 0.657 ± 0.0195051 1.441 ± 0.047 13.664 ± 0.444 0.770 ± 0.00452 1.250 ± 0.023 13.117 ± 0.243 0.769 ± 0.03753 1.315 ± 0.035 14.191 ± 0.392 0.750 ± 0.05654 1.266 ± 0.018 15.106 ± 0.231 0.825 ± 0.01055 1.207 ± 0.012 13.815 ± 0.157 0.790 ± 0.00156 1.169 ± 0.019 14.291 ± 0.270 0.776 ± 0.00557 1.285 ± 0.011 14.002 ± 0.124 0.818 ± 0.01858 1.229 ± 0.014 15.245 ± 0.157 0.769 ± 0.00659 1.228 ± 0.013 13.542 ± 0.156 0.788 ± 0.028

Table S5: Ca2+-D63NS100B 15N relaxation data, 18.8 TeslaPositio

nR1

800 R2800 NOE800

12 0.999 ± 0.036 20.185 ± 0.639 0.848 ± 0.0183 0.948 ± 0.052 18.918 ± 1.040 0.866 ± 0.0414 0.919 ± 0.039 20.508 ± 0.840 0.847 ± 0.0015 0.936 ± 0.044 20.213 ± 1.459 0.843 ± 0.0116 0.832 ± 0.015 19.926 ± 0.284 0.902 ± 0.0577 0.797 ± 0.025 19.098 ± 0.454 0.868 ± 0.0038 0.757 ± 0.022 20.862 ± 0.792 0.877 ± 0.0189 0.872 ± 0.038 18.228 ± 0.833 0.899 ± 0.03010 0.966 ± 0.031 19.861 ± 0.475 0.892 ± 0.03111 0.853 ± 0.065 20.227 ± 1.193 0.850 ± 0.05712 0.922 ± 0.088 18.918 ± 1.040 0.895 ± 0.00313 0.882 ± 0.040 24.722 ± 1.506 0.895 ± 0.06914 0.980 ± 0.056 23.229 ± 0.919 0.876 ± 0.06815 1.054 ± 0.079 20.822 ± 1.309 0.873 ± 0.04416 0.991 ± 0.023 19.473 ± 0.716 0.859 ± 0.04317 0.862 ± 0.029 19.197 ± 0.561 0.872 ± 0.04718 1.005 ± 0.043 20.201 ± 0.726 0.835 ± 0.00319 1.098 ± 0.034 20.480 ± 0.384 0.865 ± 0.03220 0.982 ± 0.013 19.638 ± 0.508 0.866 ± 0.01321 0.911 ± 0.015 18.364 ± 0.315 0.807 ± 0.01122232425 1.404 ± 0.099 21.520 ± 1.049 0.860 ± 0.00226 1.029 ± 0.022 19.147 ± 0.252 0.874 ± 0.01427 0.930 ± 0.042 20.023 ± 0.647 0.855 ± 0.04328 1.008 ± 0.046 17.231 ± 0.458 0.835 ± 0.01129 20.281 ± 1.464 0.875 ± 0.06130 0.729 ± 0.14631 1.001 ± 0.015 16.231 ± 0.212 0.863 ± 0.00732 1.074 ± 0.022 19.513 ± 0.379 0.860 ± 0.01833 0.951 ± 0.013 18.588 ± 0.410 0.872 ± 0.02134 1.025 ± 0.021 19.982 ± 0.539 0.898 ± 0.01635 0.993 ± 0.021 16.174 ± 0.304 0.874 ± 0.03336 0.941 ± 0.029 19.190 ± 0.646 0.885 ± 0.02037 1.073 ± 0.018 21.358 ± 0.682 0.881 ± 0.02138 0.965 ± 0.018 17.887 ± 0.303 0.895 ± 0.02139 0.912 ± 0.025 17.973 ± 0.545 0.859 ± 0.01840 1.004 ± 0.039 19.934 ± 0.746 0.890 ± 0.03941 1.049 ± 0.034 19.108 ± 0.759 0.849 ± 0.048

42 ±43 1.076 ± 0.097 17.326 ± 1.209 0.743 ± 0.13744 0.916 ± 0.072 20.206 ± 1.355 0.835 ± 0.00645 1.113 ± 0.101 15.061 ± 0.88946 0.957 ± 0.033 20.048 ± 1.814 0.710 ± 0.02647 1.455 ± 0.19348 0.688 ± 0.26349 1.195 ± 0.033 16.685 ± 0.457 0.573 ± 0.0195051 1.251 ± 0.049 21.167 ± 1.193 0.818 ± 0.01752 1.084 ± 0.025 19.335 ± 0.568 0.828 ± 0.01353 1.087 ± 0.047 20.158 ± 0.800 0.792 ± 0.01654 0.943 ± 0.016 19.479 ± 0.695 0.826 ± 0.00255 1.099 ± 0.013 18.307 ± 0.297 0.864 ± 0.01556 0.799 ± 0.019 18.779 ± 0.811 0.774 ± 0.01457 1.003 ± 0.010 19.932 ± 0.389 0.883 ± 0.01958 0.869 ± 0.013 21.817 ± 0.698 0.847 ± 0.00359 0.910 ± 0.014 19.362 ± 0.779 0.834 ± 0.00560 0.956 ± 0.027 18.860 ± 0.514 0.808 ± 0.0086162 0.859 ± 0.016 16.483 ± 0.408 0.755 ± 0.03063 1.001 ± 0.008 18.231 ± 0.193 0.852 ± 0.00164 1.041 ± 0.007 17.249 ± 0.114 0.869 ± 0.00965 1.013 ± 0.063 23.356 ± 1.501 0.866 ± 0.01466 0.957 ± 0.012 18.669 ± 0.466 0.876 ± 0.00167 1.020 ± 0.011 19.415 ± 0.377 0.882 ± 0.00768 0.858 ± 0.028 17.672 ± 0.366 0.886 ± 0.02769 1.106 ± 0.046 15.943 ± 0.413 0.863 ± 0.01070 1.011 ± 0.037 19.277 ± 0.468 0.887 ± 0.03371 0.976 ± 0.019 18.016 ± 0.218 0.861 ± 0.03572 0.929 ± 0.027 18.515 ± 0.562 0.878 ± 0.02673 0.976 ± 0.029 18.067 ± 0.506 0.860 ± 0.03174 1.067 ± 0.037 19.764 ± 0.434 0.858 ± 0.01275 0.995 ± 0.045 21.165 ± 0.699 0.860 ± 0.02076 0.987 ± 0.038 21.890 ± 0.535 0.868 ± 0.00577 0.984 ± 0.048 20.429 ± 0.909 0.846 ± 0.0177879 0.992 ± 0.044 20.810 ± 0.758 0.862 ± 0.03980 0.943 ± 0.010 20.231 ± 0.197 0.884 ± 0.00881 0.940 ± 0.169 22.397 ± 3.402 0.961 ± 0.0098283 1.000 ± 0.065 19.728 ± 1.228 0.819 ± 0.11484 1.191 ± 0.314 25.201 ± 2.573 0.839 ± 0.0358586 1.627 ± 0.312 0.771 ± 0.08187 22.004 ± 2.018 0.709 ± 0.01888 1.363 ± 0.056 19.464 ± 0.694 0.642 ± 0.00389 1.841 ± 0.070 16.333 ± 0.912 0.622 ± 0.00490 0.900 ± 0.031 15.963 ± 0.699 0.582 ± 0.058

91 1.686 ± 0.003 7.521 ± 0.008 0.237 ± 0.004

Table S6: TRTK-12-Ca2+-D63NS100B 15N relaxation data, 14.1 TeslaPosition R1

600 R2600 NOE600

12 1.232 ± 0.048 13.907 ± 0.715 0.835 ± 0.0133 1.092 ± 0.038 13.904 ± 0.642 0.840 ± 0.0834 1.105 ± 0.033 14.100 ± 0.539 0.868 ± 0.0475 1.146 ± 0.029 14.318 ± 0.474 0.873 ± 0.0016 1.170 ± 0.016 14.033 ± 0.249 0.856 ± 0.001789 1.118 ± 0.011 13.806 ± 0.183 0.865 ± 0.012

10 1.084 ± 0.025 14.701 ± 0.463 0.836 ± 0.03011 1.071 ± 0.024 13.810 ± 0.413 0.857 ± 0.04812 1.082 ± 0.021 14.828 ± 0.404 0.867 ± 0.00313 1.149 ± 0.019 15.077 ± 0.363 0.823 ± 0.0201415 1.060 ± 0.019 14.334 ± 0.361 0.859 ± 0.00616 1.114 ± 0.011 13.661 ± 0.173 0.896 ± 0.03017 1.116 ± 0.013 14.082 ± 0.243 0.850 ± 0.00518 1.105 ± 0.016 14.842 ± 0.300 0.827 ± 0.01719 1.374 ± 0.017 13.262 ± 0.220 0.874 ± 0.01920 1.184 ± 0.008 13.442 ± 0.142 0.858 ± 0.01221 1.106 ± 0.009 13.348 ± 0.149 0.847 ± 0.01922232425 1.425 ± 0.043 14.685 ± 0.653 0.862 ± 0.01826 1.130 ± 0.009 13.842 ± 0.166 0.861 ± 0.01327 1.240 ± 0.019 13.165 ± 0.280 0.828 ± 0.04128 1.184 ± 0.021 12.994 ± 0.293 0.886 ± 0.04129 0.806 ± 0.08530 0.912 ± 0.04731 1.126 ± 0.011 11.917 ± 0.160 0.850 ± 0.00732 1.168 ± 0.013 13.630 ± 0.204 0.840 ± 0.0033334 1.143 ± 0.011 13.759 ± 0.168 0.873 ± 0.02435 1.193 ± 0.012 12.938 ± 0.172 0.857 ± 0.01036 1.363 ± 0.032 14.266 ± 0.466 0.923 ± 0.00437 1.229 ± 0.013 14.790 ± 0.220 0.856 ± 0.01138 1.194 ± 0.012 12.849 ± 0.188 0.850 ± 0.04739 1.194 ± 0.023 12.979 ± 0.349 0.828 ± 0.0474041 1.211 ± 0.034 12.570 ± 0.508 0.815 ± 0.047

42434445 1.236 ± 0.036 14.524 ± 0.449 0.802 ± 0.04546 1.143 ± 0.009 11.493 ± 0.111 0.682 ± 0.00347 1.136 ± 0.023 12.207 ± 0.299 0.774 ± 0.00748 0.699 ± 0.10449 1.062 ± 0.096 10.123 ± 0.420 0.531 ± 0.0175051 1.253 ± 0.049 14.195 ± 0.276 0.801 ± 0.02052 1.170 ± 0.025 13.376 ± 0.176 0.825 ± 0.00353 1.091 ± 0.047 13.637 ± 0.180 0.805 ± 0.03254 1.103 ± 0.016 13.081 ± 0.193 0.853 ± 0.00255 1.081 ± 0.013 13.301 ± 0.158 0.804 ± 0.01456 1.138 ± 0.019 13.382 ± 0.180 0.844 ± 0.0085758 1.099 ± 0.007 13.680 ± 0.129 0.818 ± 0.02359 1.080 ± 0.009 12.814 ± 0.140 0.828 ± 0.00260 1.134 ± 0.011 14.351 ± 0.179 0.842 ± 0.01861 1.047 ± 0.009 13.767 ± 0.135 0.884 ± 0.03462 0.940 ± 0.009 9.329 ± 0.117 0.747 ± 0.01963 1.269 ± 0.008 11.238 ± 0.105 0.851 ± 0.01464 1.108 ± 0.009 11.701 ± 0.125 0.840 ± 0.00865 1.403 ± 0.005 14.295 ± 0.121 0.892 ± 0.03566 1.124 ± 0.012 13.186 ± 0.196 0.843 ± 0.01067 1.188 ± 0.008 14.244 ± 0.136 0.849 ± 0.01368 1.092 ± 0.016 13.257 ± 0.253 0.852 ± 0.01269 1.191 ± 0.019 13.046 ± 0.292 0.850 ± 0.02070 1.289 ± 0.020 13.818 ± 0.322 0.877 ± 0.04171 1.154 ± 0.009 12.624 ± 0.118 0.825 ± 0.01972 1.159 ± 0.018 13.578 ± 0.277 0.827 ± 0.02973 1.236 ± 0.013 13.300 ± 0.183 0.858 ± 0.00974 1.190 ± 0.013 13.597 ± 0.274 0.869 ± 0.01975 1.180 ± 0.011 13.333 ± 0.165 0.845 ± 0.03576 1.154 ± 0.012 13.131 ± 0.181 0.864 ± 0.01277 1.152 ± 0.015 13.383 ± 0.240 0.864 ± 0.04178 1.239 ± 0.013 13.932 ± 0.211 0.855 ± 0.01479 1.139 ± 0.013 13.408 ± 0.201 0.832 ± 0.01080 1.152 ± 0.015 13.716 ± 0.245 0.833 ± 0.00881 1.171 ± 0.031 15.662 ± 0.528 0.868 ± 0.00982 1.192 ± 0.021 14.581 ± 0.374 0.844 ± 0.00683 1.169 ± 0.014 13.052 ± 0.199 0.839 ± 0.00084 1.172 ± 0.017 13.359 ± 0.269 0.834 ± 0.0108586 1.322 ± 0.014 12.915 ± 0.170 0.757 ± 0.03987 1.317 ± 0.017 12.664 ± 0.198 0.765 ± 0.05088 1.343 ± 0.017 11.296 ± 0.166 0.789 ± 0.09089 1.685 ± 0.028 9.143 ± 0.192 0.643 ± 0.01590 1.540 ± 0.137 6.852 ± 0.840 0.365 ± 0.019

91 1.576 ± 0.006 4.489 ± 0.025 0.128 ± 0.003

Table S7: TRTK-12-Ca2+-D63NS100B 15N relaxation data, 18.8 TeslaPositio

nR1

800 R2800 NOE800

12 0.975 ± 0.047 20.148 ± 0.561 0.872 ± 0.0483 0.866 ± 0.061 18.453 ± 1.059 0.862 ± 0.0154 0.805 ± 0.047 21.063 ± 0.479 0.842 ± 0.0085 0.734 ± 0.035 23.055 ± 0.897 0.869 ± 0.0346 0.810 ± 0.018 18.635 ± 0.212 0.860 ± 0.025789 0.841 ± 0.015 18.971 ± 0.354 0.883 ± 0.00510 0.694 ± 0.031 20.261 ± 1.040 0.844 ± 0.01211 0.741 ± 0.033 21.053 ± 0.692 0.887 ± 0.09212 0.800 ± 0.036 22.912 ± 0.564 0.858 ± 0.00813 0.829 ± 0.037 23.924 ± 0.828 0.877 ± 0.0151415 0.803 ± 0.039 22.941 ± 0.857 0.894 ± 0.02816 0.873 ± 0.017 20.024 ± 0.453 0.854 ± 0.00317 0.855 ± 0.020 21.187 ± 0.309 0.879 ± 0.02318 0.877 ± 0.026 22.146 ± 0.469 0.867 ± 0.03119 1.190 ± 0.023 21.105 ± 0.215 0.841 ± 0.02320 0.947 ± 0.012 20.529 ± 0.380 0.859 ± 0.01321 0.921 ± 0.017 19.604 ± 0.294 0.831 ± 0.01722232425 1.308 ± 0.052 23.310 ± 0.640 0.847 ± 0.02126 0.969 ± 0.012 20.655 ± 0.185 0.866 ± 0.02227 0.868 ± 0.036 22.121 ± 0.495 0.867 ± 0.00028 0.913 ± 0.037 18.975 ± 0.360 0.847 ± 0.08729 0.855 ± 0.00130 0.863 ± 0.00731 0.919 ± 0.013 15.937 ± 0.117 0.875 ± 0.00132 0.896 ± 0.016 19.519 ± 0.186 0.842 ± 0.0043334 0.899 ± 0.014 19.058 ± 0.285 0.889 ± 0.00035 0.871 ± 0.016 18.329 ± 0.203 0.871 ± 0.04336 0.856 ± 0.019 20.945 ± 0.721 0.892 ± 0.04037 0.884 ± 0.017 22.434 ± 0.521 0.864 ± 0.03638 0.907 ± 0.017 19.798 ± 0.212 0.876 ± 0.003

39 0.861 ± 0.032 18.180 ± 0.441 0.891 ± 0.0184041 0.914 ± 0.034 15.496 ± 0.684 0.833 ± 0.07942434445 0.897 ± 0.046 18.555 ± 0.589 0.872 ± 0.04746 0.871 ± 0.012 15.566 ± 0.095 0.711 ± 0.00347 0.808 ± 0.032 21.464 ± 0.527 0.765 ± 0.01848 0.734 ± 0.01149 1.100 ± 0.025 16.758 ± 0.218 0.564 ± 0.0085051 1.093 ± 0.016 20.378 ± 0.393 0.814 ± 0.00752 0.884 ± 0.013 19.092 ± 0.250 0.817 ± 0.02053 0.809 ± 0.013 19.666 ± 0.214 0.803 ± 0.06254 0.831 ± 0.012 20.919 ± 0.309 0.886 ± 0.01755 0.855 ± 0.012 19.320 ± 0.243 0.878 ± 0.00256 0.841 ± 0.013 20.052 ± 0.362 0.871 ± 0.0055758 0.795 ± 0.009 21.190 ± 0.340 0.861 ± 0.00459 0.862 ± 0.010 18.119 ± 0.173 0.852 ± 0.00660 0.826 ± 0.017 19.726 ± 0.234 0.866 ± 0.02561 0.773 ± 0.011 20.845 ± 0.231 0.857 ± 0.00962 0.771 ± 0.012 17.067 ± 0.232 0.725 ± 0.01863 0.925 ± 0.009 19.861 ± 0.161 0.836 ± 0.00264 0.964 ± 0.009 18.071 ± 0.088 0.853 ± 0.00565 0.900 ± 0.011 19.684 ± 0.133 0.897 ± 0.00766 0.881 ± 0.012 19.431 ± 0.345 0.867 ± 0.00467 0.927 ± 0.010 20.811 ± 0.280 0.894 ± 0.01868 0.881 ± 0.028 20.798 ± 0.327 0.891 ± 0.04969 0.903 ± 0.035 16.228 ± 0.287 0.882 ± 0.02470 0.961 ± 0.030 20.779 ± 0.404 0.889 ± 0.01571 0.873 ± 0.010 18.810 ± 0.122 0.870 ± 0.04072 0.797 ± 0.023 18.759 ± 0.405 0.909 ± 0.01773 0.853 ± 0.018 19.482 ± 0.388 0.884 ± 0.00974 0.899 ± 0.020 20.926 ± 0.294 0.899 ± 0.03675 0.927 ± 0.018 23.299 ± 0.332 0.876 ± 0.00276 0.819 ± 0.020 18.182 ± 0.437 0.862 ± 0.01677 0.820 ± 0.018 19.718 ± 0.290 0.871 ± 0.02878 0.909 ± 0.022 21.766 ± 0.416 0.876 ± 0.04179 0.890 ± 0.021 19.292 ± 0.220 0.861 ± 0.01180 0.807 ± 0.017 19.897 ± 0.330 0.894 ± 0.00481 0.820 ± 0.040 24.456 ± 1.280 0.863 ± 0.03682 0.888 ± 0.036 22.522 ± 0.911 0.904 ± 0.06983 0.891 ± 0.017 18.690 ± 0.170 0.891 ± 0.02984 0.858 ± 0.023 21.092 ± 0.716 0.827 ± 0.0288586 1.040 ± 0.023 19.546 ± 0.398 0.788 ± 0.03287 0.969 ± 0.027 20.444 ± 0.646 0.768 ± 0.041

88 1.108 ± 0.028 17.981 ± 0.322 0.766 ± 0.00789 1.594 ± 0.041 15.075 ± 0.268 0.668 ± 0.02990 1.723 ± 0.113 14.707 ± 0.358 0.460 ± 0.00491 1.746 ± 0.007 7.962 ± 0.011 0.329 ± 0.004

Table S8: Optimized values of and R20 for Ca2+- D63NS100B ± TRTK-12a, b, c

Residues (Hz) R20 600 (s-1) R2

0 800 (s-1) 2/N d

Ca2+- D63NS100B9 93.19 ± 21.3 14.65 ± 0.09 17.53 ± 1.18 3.85

41 60.90 ± 12.7 14.10 ± 0.08 16.75 ± 0.13 0.0742 94.19 ± 16.4 16.64 ± 0.66 21.54 ± 0.95 0.1043 80.67 ± 9.40 14.26 ± 0.06 18.21 ± 0.08 0.7844 88.68 ± 26.4 15.33 ± 0.27 18.35 ± 0.70 0.3446 89.00 ± 31.2 12.87 ± 0.03 16.78 ± 0.19 0.3675 80.42 ± 44.9 15.23 ± 0.72 17.69 ± 0.43 0.2379 61.72 ± 18.3 14.53 ± 0.16 17.77 ± 0.50 0.1181 67.93 ± 6.10 18.87 ± 0.38 20.63 ± 0.65 0.0882 84.68 ± 28.0 16.50 ± 0.40 24.89 ± 0.38 0.0783 88.36 ± 26.2 15.90 ± 0.15 19.76 ± 0.23 0.1484 66.54 ± 10.4 15.69 ± 0.15 19.53 ± 0.32 0.1086 58.69 ± 14.0 14.20 ± 0.10 22.96 ± 0.90 0.2487 102.0 ± 17.4 14.86 ± 1.17 17.29 ± 2.20 0.16

Ca2+- D63NS100B + TRTK42 28.93 ± 0.62 22.06 ± 0.53 27.41 ± 0.99 0.1488 24.63 ± 8.43 14.92 ± 0.39 17.33 ± 0.19 0.06

a The values for and R20 were independently obtained by holding the global parameters of ex and

pa fixed to 0.880 ± 0.30 ms and 0.986 ± 0.003 for Ca2+- D63NS100B.b The values for and R2

0 were independently obtained by holding the global parameters of ex and pa fixed to 1.47 ± 0.13 ms and 0.87 ± 0.09 Ca2+- D63NS100B + TRTK-12.c The uncertainties for each value was calculated from Monte-Carlo simulations using the above ex

and pa values as initial parameters.d The 2 values listed here are normalized with respect to the number of degrees of freedom.

Table S9. Optimized relaxation dispersion parameters for the terminal Asn and Gln side chain 15N resonances for all Ca2+-S100B complexesa, b, c, d

Residues R2 eff AVE

(s-1)R2 eff RMSD

(s-1)pa ex

(10-3 s)

(Hz)R2

0 600

(s-1)R2

0 800

(s-1)2/N

WT S100BNo TRTK

Gln16 7.54 ± 0.62 1.40 ± 0.02 - - - - - -Asn37 18.8 ± 0.48 1.89 ± 0.04 0.97 ± 0.01 2.00 ± 0.65 58.6 ± 13 17.6 ± 0.50 19.4 ± 2.3 0.83 ± 0.33Asn38 13.6 ± 1.33 0.82 ± 0.03 NE NE NE NE NE NEGln50 11.3 ± 0.16 1.39 ± 0.10 0.97 ± 0.01 3.66 ± 1.51 58.6 ± 14 10.8 ± 0.15 10.8 ± 0.05 0.87 ± 0.03Gln71 6.55 ± 0.25 1.33 ± 0.11 - - - - - -

+ TRTKGln16 5.62 ± 0.53 1.43 ± 0.06 - - - - - -Asn37 21.1 ± 0.23 0.96 ± 0.01 NE NE NE NE NE NEAsn38 16.6 ± 2.34 0.77 ± 0.18 NE NE NE NE NE NEGln50 13.6 ± 0.18 1.40 ± 0.05 0.73 ± 0.20 73 ± 60 79.3 ± 10 13 ± 0.20 14 ± 0.15 1.54 ± 0.20Gln71 7.90 ± 0.50 1.33 ± 0.03 - - - - - -

D61NS100BNo TRTK

Gln16e 7.46 ± 0.89 0.88 ± 0.12 - - - - - -Asn37 11.7 ± 0.43 4.00 ± 0.04 0.98 ± 0.02 29 ± 6.20 170 ± 4.5 9.08 ± 1.10 13.6 ± 0.07 0.28 ± 0.23Asn38 9.76 ± 0.65 0.71 ± 0.25 NE NE NE NE NE NEGln50 5.85 ± 0.03 3.31 ± 0.04 - - - - - -Asn61 11.6 ± 0.75 1.91 ± 0.36 0.98 ± 0.01 4.69 ± 1.33 134 ± 39 10.4 ± 1.05 17.7 ± 1.05 0.66 ± 0.14Gln71e 7.05 ± 0.44 1.37 ± 0.04 - - - - - -+ TRTKGln16e 9.12 ± 0.97 0.80 ± 0.06 - - - - - -Asn37 19.2 ± 2.06 0.83 ± 0.07 NE NE NE NE NE NEAsn38 11.6 ± 1.56 0.50 ± 0.13 NE NE NE NE NE NEGln50 10.3 ± 0.17 1.75 ± 0.37 0.98 ± 0.003 3.78 ± 0.29 51.3 ± 5.9 9.71 ± 0.06 12.0 ± 0.28 1.87 ± 0.45Asn61 28.0 ± 1.81 2.33 ± 0.23 0.97 ± 0.03 0.13 ± 0.09 531 ± 282 20 ± 1.00 22.9 ± 1.90 1.30 ± 0.07Gln71e 7.03 ± 0.97 0.88 ± 0.07 - - - - - -

D63NS100BNo TRTK

Gln16 5.62 ± 0.53 1.43 ± 0.06 - - - - - -Asn37 14.7 ± 0.09 1.36 ± 0.04 0.97 ± 0.02 5.56 ± 2.53 49.5 ± 4.5 14.3 ± 0.20 18.9 ± 0.85 0.36 ± 0.12Asn38 11.7 ± 0.27 0.82 ± 0.03 NE NE NE NE NE NEGln50 9.82 ± 0.07 1.38 ± 0.11 0.94 ± 0.04 5.30 ± 4.42 84.5 ± 14 8.65 ± 0.63 10.7 ± 0.15 0.80 ± 0.05Asn63 19.0 ± 1.88 1.82 ± 0.42 0.76 ± 0.16 57.5 ± 39 90.2 ± 40 18.3 ± 1.5 21.9 ± 1.9 1.98 ± 0.00Gln71 4.14 ± 0.17 1.30 ± 0.12 - - - - - -

+ TRTKGln16 7.31 ± 0.69 1.37 ± 0.09 - - - - - -Asn37 17.8 ± 0.64 0.89 ± 0.21 NE NE NE NE NE NEAsn38 15.3 ± 0.79 0.55 ± 0.22 NE NE NE NE NE NEGln50 10.8 ± 0.08 1.40 ± 0.09 0.98 ± 0.01 62 ± 5.9 239 ± 86 8.65 ± 0.63 9.36 ± 1.3 1.07 ± 0.17Asn63 21.4 ± 1.29 0.92 ± 0.06 NE NE NE NE NE NEGln71 4.90 ± 0.46 1.30 ± 0.13 - - - - - -

D65NS100BNo TRTK

Gln16 e 12.2 ± 3.01 3.59 ± 0.08 - - - - - -Asn37 EB EB EB EB EB EB EB EBAsn38 2.22 ± 0.60 16.0 ± 1.25 0.93 ± 0.06 2.06 ± 1.31 100 ± 58 14.2 ± 1.20 23.5 ± 2.10 0.81 ± 0.22Gln50 1.89 ± 0.05 13.6 ± 0.94 0.91 ± 0.03 1.06 ± 0.07 36.5 ± 6.5 13.0 ± 0.40 15.4 ± 0.20 1.14 ± 0.20Asn65 EB EB EB EB EB EB EB EBGln71 e 9.00 ± 0.89 2.64 ± 0.04 - - - - -+ TRTKGln16 e 9.90 ± 0.88 0.77 ± 0.14 - - - - - -Asn37 20.8 ± 0.71 0.44 ± 0.10 NE NE NE NE NE NEAsn38 12.2 ± 1.79 0.87 ± 0.41 NE NE NE NE NE NEGln50 10.9 ± 0.08 0.87 ± 0.13 NE NE NE NE NE NEAsn65 EB EB EB EB EB EB EB EBGln71 e 6.68 ± 0.80 0.84 ± 0.07 - - - - - -

a The average (AVE) and RMSD values for R2 eff are given for 14.1 T.

Fig. S1 HSQC of the D61NS100B mutant in the presence (red) and absence (black) of fully

saturated TRTK-12 peptide. Experiments were collected at 37C, pH 7.2 on a 600 MHz

NMR spectrometer.

Fig. S2 HSQC of the D63NS100B mutant in the presence (red) and absence (black) of fully

saturated TRTK-12 peptide. Experiments were collected at 37C, pH 7.2 on a 600 MHz

NMR spectrometer.

Fig. S3 HSQC of the D65NS100B mutant in the presence (red) and absence (black) of fully

saturated TRTK-12 peptide. Experiments were collected at 37C, pH 7.2 on a 600 MHz

NMR spectrometer.

Fig. S5 The R.M.S.D of measured R2 eff values for backbone amide residues of Ca2+-

S100B in the presence (red) and absence (black) of TRTK-12 collected at 14.1 T. The

red dotted line represents values 2x’s greater than the median of the rmsd R2 eff values.

In the presence of peptide, the average R2 eff values are significantly lower in areas

corresponding to the S100B-peptide binding interface (i.e. hinge region and C-terminus).

Fig. S6 Selected relaxation dispersion curves of Apo-S100B ± TRTK-12 peptide.

Dispersion profiles of the backbone amides of K55 and G66 in the absence and

presence of TRTK-12 are shown with data collected at 800 (blue) and 600 (black) MHz.

(b) Evidence for chemical exchange for Apo S100B due to broadened/missing peaks

(black), model-free (red), and T1/T2 vs DNH (green). (c) Residues highlighted in red have

evidence for chemical exchange.

Fig. S7 Schematic illustrating states of S100B that are detected thus far in

thermodynamic/kinetic landscape via structural and protein dynamic information

(NMR/Xray). The states illustrated represent those significantly populated (>1%). The

triangle represents the “closed” structure of S100B with helix 3 parallel to helix 4 for EF2

and the circle represents the “open” conformation of S100B in which helix 3 is

perpendicular to helix 4 in EF2 (Fig. 2). The dashed circles represent “dynamic” states

of S100B for which NMR has detected significant populations that may affect binding

kinetics and thermodynamics. Targets (T) include TRTK-12, p53, hdm2, and hdm4.

Target binding and functional mini-folding event (“TBFF”), where S100B does not

achieve its final structure unless bound to a target (i.e extension of helix 4). Note –

unlike some EF-hand proteins, an “open” conformation of S100B has not yet been

detected (<1%; Fig. S6), so this state is not included here.