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Coulomb interactions between internal ionizable groups and surface charges in proteins

Coulomb interactions between internal ionizable groups and surface charges in proteins. Victor Khangulov Dec 14, 2009 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno.

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Coulomb interactions between internal ionizable groups and surface charges in proteins

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  1. Coulomb interactions between internal ionizable groups and surface charges in proteins Victor Khangulov Dec 14, 2009 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno

  2. Internal ionizable groups govern many biochemical processes • Photoactivation • Ion homeostasis • H+ transport • e- transfer • Catalysis Most ionizable residues are located on the protein surface. Internal ionizable residues are responsible for energy transduction:

  3. Apparent pKa values of Lys at 25 internal positions Normal pKa of Lys in water Isom, Daniel Gerard.  pKa values of internal ionizable groups in staphylococcal nuclease [Ph.D. dissertation]. United States -- Maryland: The Johns Hopkins University; 2006.

  4. Measurement of pKa values: Ideal CaseTitration of Lys-25 inL25K Background L25K • Assumption: Group behaves independently • We know pKa values of all H,D, and E • His-8 pKa = 6.3 • His-121 pKa = 5.4 • Asp-21 pKa = 6.5 • Everything else titrates ≤ 4.5 • For “ideal” cases, H8, H121 and D21 are not affected by internal lysine. Figure from D. Isom

  5. Measurement of pKa value: Non-Ideal CaseTitration of Lys-62 inT62K Background T62K His-8, His-121 or Asp-21 potentially are affected by the ionization of Lys-62 Figure from D. Isom

  6. Titration of His residues is not linked to ionization of Lys-62 Data from C. Castañeda

  7. Lys-62 is buried and 6.3 Å away from Asp-21 T62K (PHS) PDB: 3DMU Resolution: 1.80Å R-work: 0.198 R-free: 0.255

  8. pKa of Lys-62 shifts down in D21N variant Background Background T62K D21N/T62K pKa = 7.0 ± 0.2 pKa = 8.1 ± 0.1

  9. NMR confirms pKa of Lys-62 obtained through linkage analysis T62K ∆(1HN) Chemical Shift (ppm) pKa = 8.10 ± 0.01 (Linkage pKa = 8.1 ± 0.1) pH

  10. Lys-62 pKa shifts further down in D21N variant D21N/T62K ∆(1HN) Chemical Shift (ppm) pKa = 6.71 ± 0.03 (Linkage pKa = 7.0 ± 0.2) pH

  11. Lys-62 pKa is also influenced by Asp-19 D19N/T62K ∆(1HN) Chemical Shift (ppm) pKa = 7.43 ± 0.05 pH

  12. Effect of Asp-21 on pKa of Lys-62 T62K ∆+PHS pKa2 = 8.1 pKa1 = 6.7 D21N D21N/T62K ∆Gcoupling = 1.36 (pKa2 – pKa1) = 1.36 (8.10 – 6.71 ) = 1.89 kcal/mol (±0.03)

  13. Titration of other groups in the presence of Lys-62 is not affected T62K T62K ∆+PHS ∆+PHS D19 D21 T62K ∆+PHS ∆+PHS T62K E67 E43

  14. Titration of Asp-21 shows dependence on the presence of Lys-62 Asp-21 in T62K pKa = 4.17 ± 0.08 n = 1.14 ± 0.13 Asp-21 in ∆+PHS pKa = 6.54 ± 0.02 n = 2.00 ± 0.02

  15. Effect of Lys-62 on the pKa of Asp-21 T62K ∆+PHS pKa1 = 4.1 pKa2 = 6.6 D21N D21N/T62K ∆Gcoupling = 1.36 (pKa2 – pKa1) = 1.36 (6.54 – 4.17) = 3.22 kcal/mol (±0.08)

  16. ∆Gij is not symmetric between Asp-21 and Lys-62 T62K ∆+PHS pKa2 = 8.1 pKa1 = 4.1 pKa2 = 6.6 pKa1 = 6.7 This is the best estimate of ∆Gcoupling D21N D21N/T62K ∆Gcoupling (Lys-62) = 1.88 ± 0.03kcal/mol ∆Gcoupling (Asp-21) = 3.22 ± 0.08 kcal/mol

  17. The magnitude of the coupling between internal and surface ionizable groups could be governed by other factors Electronic polarization Bulk water Fixed permanent dipoles Water penetration Relaxation of permanent dipoles Second internal charge Surface charges Local unfolding Fluctuations of surface charges Global unfolding

  18. Native structure is not perturbed by neutral Lys-62 Background (PHS) PDB: 1EY8 Resolution: 1.75Å R-work: 0.187 R-free: 0.240 T62K (PHS) PDB: 3DMU Resolution: 1.80Å R-work: 0.198 R-free: 0.255

  19. Structure (as reported by TALOS+) is not perturbed by charged Lys ∆+PHS at pH 4.66 T62K at pH 4.52 Chemical Shifts: (C,CA,H,HN,HA) Difference of > 30° in φ or ψ Between ∆+PHS and T62K T62K residues in intermediate Exchange.

  20. Summary • There is a strong interaction between the internal Lys-62 and Asp-21. • There is minor interaction between Lys-62 and Asp-19. • The pKa values of all other Asp and Glu residues are not affected by the ionization of Lys-62. • The ionization of Lys-62 does not trigger detectable conformational reorganization.

  21. 9 Internal Lys Variants Show Evidence of Coupling to another ionizable Residue T62K

  22. Crystal Structures F34K L36K V23K PDB: 3ITP PDB: 3EJI Synchrotron Data A90K V104K L103K T62K PDB: 3E5S PDB: 3C1F Synchrotron Data PDB: 3DMU

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