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Interactions of Lys-104 and Lys-34: Research Update

This research update explores the interactions of Lys-104 and Lys-34 with a network of charges in biological interactions. It discusses the coupling of ionizations and the impact on neighboring residues. The study investigates titration values, pKa values, and crystal structures to understand the interconnectedness of these ionizable residues. The data collected from titrations, crystal structures, and HSQC spectra provide valuable insights for further investigation into the ionization of Lys-104 and Lys-34 and their effects on surrounding residues.

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Interactions of Lys-104 and Lys-34: Research Update

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  1. DOUBLE FEATURE F34K V104K

  2. Research Update:Interactions of Lys-104 and Lys-34 with a network of charges Victor Khangulov November 15, 2010 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno

  3. 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

  4. 9 Non-Ideal CasesTitration of Lys-62 inT62K Background T62K One or more ionizable groups are being affected by the ionization of Lys-62. Figure from D. Isom

  5. 9 Internal Lys Variants Show Evidence of Coupling to another ionizable Residue F34K V104K

  6. Ionization of Lys-104 is coupled to the titration one or more residues Reference Lys-104 V104K Lys-104 pKa value = 7.7 ± 0.1 PDB ID: 3C1F Data from Dan Isom

  7. Residues affected by Lys-104 (CBCGCO) ∆+PHS - Black ∆+PHS V104K - Red Glu-101 Glu-122 Glu-129

  8. Lys-104 is located in the vicinity of a number of Asp and Glu residues

  9. The pKa values of Asp-21 and Glu-122 are affected not only by Lys-104 but by other neighboring Glus Asp-21 Glu-122 pKa2 = 7.87 ± 0.05 pKa2 = 7.42 ± 0.01 pKa1 = 3.93 ± 0.01 pKa1 = 4.04 ± 0.03 pKa = 6.56 ± 0.01 pKa = 6.82 ± 0.04 pKa = 3.99 ± 0.02

  10. Titrations of Glu-101 and Glu-129 also reflect a network of interactions involving Lys-104 and Glus nearby Glu-101 Glu-129 pKa2 = 7.87 ± 0.05 pKa2 = 6.81 ± 0.11 pKa2 = 7.60 ± 0.08 pKa2 = 7.04 ± 0.60 pKa = 3.85 ± 0.01 pKa = 3.93 ± 0.01 pKa1 = 3.56 ± 0.02 pKa1 = 3.80 ± 0.01 pKa1 = 3.57 ± 0.05 pKa1 = 3.83 ± 0.02

  11. His are not affected by Lys-104

  12. CBCGCO Titrations ∆+PHS - Black ∆+PHS V104K – Red ∆+PHS D21N/E101Q/V104K – Blue ∆+PHS D21N/V104K/E122Q – Green

  13. HSQC spectra show multiple minor transitions possibly reflecting titrations of His-8, His-121, Lys-104, and Asp-21 ∆+PHS - Black ∆+PHS V104K – Red ∆+PHS D21N/V104K – Blue

  14. Titration of Lys-104 is coupled to the network of other ionizable residues

  15. Data Collected: CBCGCO/HSQC Titrations: V104K D21N/E101Q/V104K D21N/V104K/E122Q His pKa V104K Assignments: D21N/V104K ddG vs. pH V104K Crystal Structures V104K (3C1F) Data Needed: CBCGCO/HSQC Titrations: D21N/V104K/E129Q HSQC Titration (Lys-104 pKa from global fit) D21N/V104K/Triple mutant More Assignments? What do I have and what do I need to investigate Lys-104?

  16. Ionization of Lys-34 is coupled to the titration one or more residues Reference Lys-34 F34K Lys-34 pKa value = 7.1 ± 0.1 PDB ID: 3ITP Data from Dan Isom

  17. Residues affected by Lys-34 (CBCGCO) ∆+PHS - Black ∆+PHS F34K – Red

  18. Titration of Asp-77 is dramatically affected by Lys-34 Asp-77 Asp-83 pKa = 6.00 ± 0.05 pKa = 5.69 ± 0.04

  19. Ionization of Lys-34 affects pKa values of neighboring Glu residues Glu-10 Glu-122 pKa2 = 6.60 ± 0.37 pKa = 2.92 ± 0.01 pKa1 = 3.75 ± 0.04 pKa = 3.64 ± 0.05 pKa = 3.99 ± 0.02

  20. HSQC spectra show multiple minor transitions possibly reflecting titrations of His-8, His-121, Lys-34 and Tyrosines

  21. Ionization of Lys-34 perturbs the titrations of Asp and Glus in a specific region of the protein

  22. Lys-34 is interacting with a network of charges

  23. Data Collected: CBCGCO/HSQC Titrations: D21N/F34K ddG vs. pH F34K D21N/F34K (Lys-34 pKa ≈ 6.5) Crystal Structures F34K (3ITP) Data Needed: CBCGCO/HSQC Titrations: F34K His pKa values F34K Assignments F34K What do I have and what do I need to investigate Lys-34?

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