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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 August 31, 2011 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 August 31, 2011 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno

  2. Internal ionizable groups in proteins 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. Can we engineer useful enzymes? - + • Structure-based Calculations: • Overestimate electrostatic energies • Underestimate protein polarizability

  4. Background • pKa values • Dielectric constants • Factors that determine pKa values of internal ionizable groups • Measurement of pKa values of internal ionizable groups • pKa values in SNase?

  5. Shift in a pKa value is related to the Gibbs free energy (ΔG°) + ΔG° total = 1.36 (10.4 – 8.0) = 3.2 (kcal/mol) pKa = 10.4 pKa = 8.0

  6. Self and Coulomb Energies Coulomb Energy Self Energy - - + ri,j ε ε + + + + + εout εin εout εin

  7. What is a dielectric constant (ε)? A macroscopic parameter that describes the polarizability of a material E 80 Water ε - 70 + - + - + 60 Liquid amides - + - + - + 50 DMSO - + - + 40 - + - + 30 - + - + 20 - + - + Solid amides - + - 10 + Dry protein powder - + - + Vacuum (ε = 1) 0 Parallel plate capacitor Dielectric Constants of Materials (2011). Clipper Controls

  8. Dependence of Coulomb energy on the dielectric constant (εp) - ri,j= 12 Å ri,j= 3 Å ri,j + ε

  9. pKa values of internal ionizable groups: Ideal Case Titration of Lys-25 in L25K variant in staphylococcal nuclease Background L25K Isom DG (2011). PNAS

  10. A systematic study of pKa values of internal ionizable residues in staphylococcalnuclease Isom DG (2011). PNAS

  11. pKa values of internal groups are anomalous Asp, Glu, and Lys at 25 internal positions in SNase Isom DG (2011). PNAS

  12. Measurement of pKa values: Ideal Case Background • Assumption: Group behaves independently • In “ideal” case, internal Lys does not affect pKa values of other ionizable groups L25K Isom DG (2011). PNAS

  13. Measurement of pKa values: Non-Ideal Case Lys-62 Lys-25 Background Background T62K L25K Isom DG (2011). PNAS

  14. Goals of this study • Determine the extent to which surface charges can be sensed inside a protein • Examine the effect of surface charges on pKa values of internal ionizable groups • Determine dielectric properties that govern communication between internal and surface charges in proteins

  15. Variants of SNase where internal Lys is coupled to other ionizable groups Isom DG (2011). PNAS

  16. Factors that can affect pKa values in proteins

  17. Coupling between internal and surface ionizable residues Shift in the pKa of one Lys only (Lys pKaD = 10.4) Background T62K pKaN = 8

  18. Coupling between internal and surface ionizable residues Shift in the pKa of Lys and His (Lys pKaD = 10.4, His pKaD = 6) Background T62K His pKa,2N shifted down

  19. Interactions between Lys-62 and surface residues • What is the microenvironment of Lys-62? • Which groups are interacting with Lys-62? • What is the magnitude of these interactions? • What are the structural consequences of ionization of Lys-62? • Can these interactions be reproduced self-consistently with continuum electrostatics calculations?

  20. Lys-62 is buried , 6.3 Å from Asp-21 and 8.4 Å from Asp-19 T62K (PHS) PDB: 3DMU Resolution: 1.80Å R-work: 0.198 R-free: 0.255

  21. D21N Substitution Affects pKa of Lys-62 Reference T62K His-8 His-121 Lys-62 pKa = 8.1  0.1 Isom DG (2011). PNAS

  22. D21N Substitution Affects pKa of Lys-62 D21N Reference D21N/T62K T62K Lys-62 pKa = 6.8 0.1 Lys-62 pKa = 8.1  0.1 Isom DG (2011). PNAS

  23. Depressed pKa of Lys-62 confirmed by NMR spectroscopy T62K ∆(1HN) Chemical Shift (ppm) pKa = 8.10 ± 0.01 (pKa= 8.1 ± 0.1 by linkage analysis)

  24. pKaof Lys-62 is depressed further in the D21N variant of SNase D21N/T62K ∆(1HN) Chemical Shift (ppm) pKa = 6.71 ± 0.03 (pKa= 6.8 ± 0.1 by linkage analysis) pH

  25. Lys-62 reports on strong interaction with Asp-21 (1.9 kcal/mol) - - pKa2 = 8.1 + + Coupling between Lys-62 and Asp-21 ∆G = 1.36 (pKa2 – pKa1) = 1.36 (8.1 – 6.7 ) = 1.9 kcal/mol pKa1 = 6.7 +

  26. CBCGCOexperiments probe the side chain carboxylic groups directly

  27. 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 reference protein pKa = 6.56 ± 0.02 n = 2.00 ± 0.02 Castañeda CA (2009). Proteins

  28. Asp-21 reports on strong interaction with Lys-62 (3.3 kcal/mol) - - + + Coupling between Lys-62 and Asp-21 pKa1 = 4.1 pKa2 = 6.6 ∆G = 1.36 (pKa2 – pKa1) = 1.36 (6.6 – 4.2 ) = 3.3 kcal/mol +

  29. Asymmetry in ∆Gijreported by Asp-21 and Lys-62 - - pKa2 = 8.1 + + Coupling between Lys-62 and Asp-21 pKa1 = 4.1 pKa2 = 6.6 ∆Gi,j (Lys-62) = 1.9 kcal/mol ∆Gi,j (Asp-21) = 3.3 kcal/mol pKa1 = 6.7 +

  30. Which ∆Gijcorrectly reflects coupling between Lys-62 and Asp-21? Lys-62 Asp-21 Lys Asp/Glu His ∆Gi,j (Asp-21) = 3.3 kcal/mol ∆Gi,j (Lys-62) = 1.9 kcal/mol

  31. What other pKa values are affected by Lys-62? ΔpKa between reference and variant protein with Lys-62

  32. Is the interaction between Lys-62 and Asp-21 through protein or through solvent? 100 mM Salt 1M Salt Asp-21 in T62K pKa = 4.2 - - pKa 4.2 • Interaction not shielded by high salt • pKa of Asp-21 remains low Case 1:Through Protein interaction + + - - • Charge-charge interaction is shielded by high salt. • pKa of Asp-21 returns to its “normal” value in the reference protein pKa = 4.2 pKa 6.1 Case 2:Through Solvent interaction + +

  33. Is the interaction between Lys-62 and Asp-21 through protein or through solvent? 1M Salt - 100 mM Salt 6.1 6.6 pKa of Asp-21 in Reference protein + pKa of Asp-21 in T62K 3.8 4.2 Lys-62 interacts with Asp-21 through protein

  34. Native structure is not perturbed by neutral Lys-62 in the crystal structure 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

  35. 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 background and T62K T62K residues in intermediate Exchange.

  36. Can structure-based calculations reproduce pKavalues of Lys-62? Lys-62 experimental pKa values pKa in T62K: 8.1 pKa in D19N/T62K: 7.5 pKa in D21N/T62K: 6.7 Dielectric constant of 10.3 to11.5 reproduced pKa of Lys-62. FDPB (UHBD), FULL/PARSE, Probe =1.4Å, Stern layer = 2.0Å Fitch CA (2002). Biophysical Journal Karp DA (2007). Biophysical Journal

  37. Structure-based continuum calculations cannot reproduce pKa values and interactions Calculated ΔGi,jvalue between Lys-62 and Asp-19 and Asp-21 ΔGi,j(Asp-21 to Lys-62) = 1.9 kcal/mol εin required to reproduce ΔGi,j 6.7 (Asp-21 with Lys-62) > 9.5 (Asp-19 with Lys-62) ΔGi,j(Asp-19 to Lys-62) = 0.8 kcal/mol

  38. Conclusions • Most pKa values are unaffected by Lys-62. • There is a strong interaction between the internal Lys-62 and Asp-21. • There is a weaker (but still strong) interaction between Lys-62 and Asp-19. • The interaction between Lys-62 and Asp-21 appears to be through protein • Therefore, the interaction between Lys-62 and Asp-21 is predominantly Coulomb in nature • pKa and ΔGij cannot be reproduced with the same dielectric constant

  39. Variants of SNase that where internal Lys titration is linked to other ionizable groups pKa = 7.1 pKa = 7.2 pKa = 7.7 Isom DG (2011). PNAS

  40. Internal Lys-34, Lys-36, and Lys-104 do not perturb the protein structure Reference (3BDC) F34K (3ITP) L36K (3EJI) L104K (3C1F)

  41. Lys-34, 36, and 104 are buried in very different environments F34K (3ITP) L36K (3EJI) L104K (3C1F) Residues within 3.5 Å of buried Lys Residues within 6.4 Å of buried Lys

  42. Titration of His was virtually insensitive to the presence of internal charged Lys His-8 His-121 • Reference •  F34K •  L36K •  V104K Largest shift of 0.3 pKa units was observed for His-121 in F34K variant of SNase

  43. Lys-34 has a weak interaction with Asp-21 Lys-34 and Asp-21 are 14 Å apart Reference F34K Lys-34 with Asp-21 pKa = 7.1 ± 0.1

  44. Lys-34 has a weak interaction with Asp-21 D21N Lys-34 and Asp-21 are 14 Å apart D21N/F34K Lys-34 with Asp-21 pKa = 7.1 ± 0.1 0.8 kcal/mol Lys-34 w/o Asp-21 pKa = 6.5 ± 0.6

  45. Titrations of Asp and Glu residues in the F34K variant  Reference  F34K  D21N/F34K • Glu-10, Asp-77, Asp-82 and Glu-122 report on weak interactions with Lys-34 • Titration of Glu-122 may be reporting on the pKa of Lys-34 in D21N/F34K variant • Since changes are globally distributed, interaction cannot be determined to be exclusively Coulomb in nature pKa = 6.60 ± 0.37

  46. What about other pKavalues in F34K? ΔpKa between reference and variant protein with Lys-34

  47. Ionization of Lys-34 contributes to globally distributed changes in the HSQC Peaks not assigned Peak broadening Changes in Shifts > 1ppm Chemical shifts were compared 0.5 pH units Above and below pKa of Lys-34 (7.1)

  48. Lys-36 exhibits high complexity in the ΔΔG vs. pH data Lys-36 and Asp-21 are 6.3 Å apart Reference L36K Lys-36 in L36K pKa = 7.2 ± 0.1

  49. pKa values of Asp, Glu and His are not perturbed in L36K ΔpKa between reference and variant protein with Lys-36

  50. Lys-36 exhibits high complexity in the ΔΔG vs. pH data Lys-36 and Asp-21 are 6.3 Å apart D21N D21N/L36K Lys-36 in L36K pKa = 7.2 ± 0.1 2.3 kcal/mol Lys-36 in D21N/L36K pKa= 5.5 ± 1.1

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