Structural origins of high apparent dielectric constants experienced by ionizable groups in the hydrophobic core of a protein

Bertrand García-Moreno, Michael S. Chimenti, Carlos A. Castañeda, Ananya Majumdar

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pKa values of 5.7, 8.8, and 8.9, respectively. To reproduce these pKa values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pKa calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.

Original languageEnglish (US)
Pages (from-to)361-377
Number of pages17
JournalJournal of Molecular Biology
Issue number2
StatePublished - Jan 14 2011
Externally publishedYes


  • NMR
  • conformational reorganization
  • dielectric constant
  • electrostatics

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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