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Research Update: Lys with Weak Interactions with Surface Charges

Research Update: Lys with Weak Interactions with Surface Charges. Victor Khangulov February 28, 2011 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno. Electronic polarization. Bulk water. Fixed permanent dipoles.

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Research Update: Lys with Weak Interactions with Surface Charges

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  1. Research Update:Lys with Weak Interactions with Surface Charges Victor Khangulov February 28, 2011 Institute in Multiscale Modeling of Biological interactions Johns Hopkins University Laboratory of Dr. Garcia-Moreno

  2. 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 What are possible molecular determinants of pKa values of internal Lys residues?

  3. Measurement of pKa values (L25K):Lys without interactions with surface charges • Assumption: Group behaves independently • We know pKa values of all H,D, and E in the reference protein ∆+PHS • 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. Background L25K Figure from D. Isom

  4. Measurement of pKa values (T62K):Lys with interactions with surface charges Background One or more ionizable groups are being affected by the ionization of Lys-62. T62K Figure from D. Isom

  5. 9 Internal Lys Variants Show Evidence of Coupling to another ionizable Residue Figure from D. Isom

  6. Internal Lys pKa values are hard to resolve with Equil. Thermo. due to high stability of D21N Background D21N D21N V104K ∆+PHS ∆+PHS L103K D21N/F34K F34K D21N/L103K two-site pKa = 8.23 ± 0.11 single-site pKa = 7.10 ± 0.12 pKa = 8.05 ± 0.21 pKa = 6.52 ± 0.14 (single-site) pKa = 7.54 ± 0.12

  7. His are not significantly affected by ionization of internal Lys V104K F34K

  8. Weak interactions between internal Lys-104 and Surface Carboxylic Groups

  9. Weak interactions between internal Lys-34 and Surface Carboxylic Groups

  10. Weak interactions between Internal Lys Affects pKa values of many Carboxylic Groups Simultaneously

  11. Extent and Distribution of Effects of Weak Interactions of the Internal Lys Vary with Position V104K L36K T41K F34K Broadening of amide chemical shift due to Lys ionization Amide chemical shift changes > 1ppm due to Lys ionization Position of internal Lys

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