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DOUBLE FEATURE

DOUBLE FEATURE. F34K. V104K. 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.

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DOUBLE FEATURE

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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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