Ionizable groups that are sequestered from water in the protein interior play critical roles in energy transduction processes. Generally, the protein interior has lower polarizability and polarity than bulk water; therefore, these groups experience potentially large electrostatic energies including dehydration, polar interactions, and Coulomb interactions. A quantitative understanding of the structure-function relationship of proteins with internal ionizable groups requires a complete description of the energetic terms experienced by these internal groups. To date, computational models of electrostatics in the protein interior fail to reproduce these energetic terms accurately. To better understand the relationship between charge burial and energy, the given energetic terms must be measured in a well-controlled experimental system. This thesis describes three investigations of this sort. The general approach is to substitute ionizable residues at internal positions in Staphylococcal nuclease (SNase), measure the pKa values of the internal groups, then to separate the relevant factors that determine the measured pKa values. The first chapter describes the problem, reviews current attempts to model protein electrostatics, and outlines the probable reasons for the failure of these models. The second chapter describes an investigation of the electrostatics of internal Arg residues. The pKa values of Arg were found to be much less shifted than those of Lys at the same positions in SNase. Crystal structures of three of the Arg-containing SNase variants suggest that this is because the Arg side chain can form multiple hydrogen bonds to polar protein atoms and site-bound water molecules. The third and fourth chapters describe detailed investigations of the determinants of the pKa values of Lys, Glu, and Asp substituted at position 38 in SNase. The p Ka value of Lys-38 is apparently normal, whereas the p Ka values of Glu-38 and Asp-38 shifted toward the neutral state by 2.4 and 3.2 units, respectively. This is because Lys-38 induces structural reorganization and is hydrated in solution. Glu-38 and Asp-38, on the other hand, are part of a hydrogen-bond network: a balance of dehydration, favorable polar interactions, and unfavorable Coulomb interactions determines their pKa values. Overall, these results illustrate the complex interplay between the ionization state of the internal residue, local stability, and specific electrostatics interactions. Further, these results provide a set of quantitative constraints that can be used to verify that structure-based electrostatics calculations reproduce experimental p Ka values for the correct physical reasons.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 69/12, Dissertation Abstracts International|
|Keywords:||Electrostatics, Internal ionizable groups, Staphylococcal nuclease|
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