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1 Approaches to the Assignment of 19 F Resonances from 3-Fluorophenylalanine Labeled Calmodulin using Solution State NMR Journal of Biomolecular NMR Julianne L. Kitevski-LeBlanc, 1 Ferenc Evanics, 1 R. Scott Prosser 1,2* 1 Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, Ontario, Canada, L5L 1C6. 2 Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 * Corresponding scott.prosser@utoronto.ca Supplementary Material 1

2 Figure S1. NMR-based assignment strategy for phenylalanine 12. A) 1 H- 19 F HSQC spectrum of 3-FPhe TR1C with 19 F 1D trace along the indirect detect fluorine axis. The δ proton crosspeak is indicated for F12. B) 1 H- 19 F NOESY-HSQC spectrum. The the beta proton of F12. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of lysine 13. All experiments were carried out at 37 C. 2

3 Figure S2. NMR-based assignment strategy for phenylalanine 16. A) 1 H- 19 F HSQC spectrum of 3-FPhe TR1C with 19 F 1D trace along the indirect detect fluorine axis. The ε, δ, and ζ proton crosspeaks are indicated for F16. B) 1 H- 19 F NOESY-HSQC spectrum. The experiment was collected as a 2D (i.e. without evolving the 19 F dimension) and shown are the first indirect and direct detect dimensions. The crosspeak highlighted corresponds to the beta protons of F16. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of serine 17. All 3

4 Figure S3. NMR-based assignment strategy for phenylalanine 19. A) 1 H- 19 F HSQC spectrum of 3-FPhe TR1C with 19 F 1D trace along the indirect detect fluorine axis. The δ and ζ proton crosspeaks are indicated for F19. B) 1 H- 19 F NOESY-HSQC spectrum. The the beta protons of F19. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of aspartic acid 20. All 4

5 Figure S4. NMR-based assignment strategy for phenylalanine 68. A) 1 H- 19 F HSQC spectrum of 3-FPhe TR1C with 19 F 1D along the indirect detect fluorine axis. The ε, δ, and ζ proton crosspeaks are indicated for F68. B) 1 H- 19 F NOESY-HSQC spectrum. The the beta protons of F68. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of leucine 69. All 5

6 Figure S5. NMR-based assignment strategy for phenylalanine 89. A) 1 H- 19 F HSQC spectrum of 3-FPhe CaM with 19 F 1D trace along the indirect detect fluorine axis. The δ and ζ proton crosspeaks are indicated for F89. B) 1 H- 19 F NOESY-HSQC spectrum. The the beta protons of F89. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of arginine 90. All 6

7 Figure S6. NMR-based assignment strategy for phenylalanine 141. A) 1 H- 19 F HSQC spectrum of 3-FPhe CaM with 19 F 1D trace along the indirect detect fluorine axis. The δ proton crosspeak is indicated for F141. B) 1 H- 19 F NOESY-HSQC spectrum. The the beta protons of F141. C) 1 H- 15 N NOESY-HSQC spectrum. All three dimensions of the experiment were collected. Shown is the nitrogen plane corresponding to ppm. The crosspeak indicated corresponds to the amide proton of valine 142. All NOTE: Residue F65 preceeds a proline residue, and therefore cannot be assigned using this method. 7

Protein Structure and Function! Lecture 4: ph, pka and pi!

Protein Structure and Function! Lecture 4: ph, pka and pi! Definition of ph and pk a! ph is a measure of the concentration of H +.! + ph = log 10[H ] For a weak acid,! HA #!!"! H + + A!, K a = [H + ][A!