Identification and Characterization of Buried Ionizable Groups in Proteins

Abstract

Ionizable groups buried in the hydrophobic interior of proteins play many essential biological roles. their physical properties are not well understood. Owing to the inherent differences in the dielectric properties of proteins and of water the properties of internal and surface ionizable groups can be very different. In a previous systematic study from this laboratory Asp, Glu, Lys and Arg residues were buried at 25 internal locations in staphylococcal nuclease. Most of these internal residues titrate with highly anomalous pKa values. Many crystal structures of these variants show that the majority of these buried ionizable residues are buried completely and in very diverse microenvironments. A survey of the properties and microenvironments of naturally occurring internal ionizable groups in proteins was undertaken with a large set of proteins from the Protein Data Bank to compare against the internal ionizable groups buried artificially in nuclease. Internal ionizable groups were identified based on accessible surface area and depth of burial using a self-consistent definition of the interface between protein and bulk water. For purposes of this survey, internal ionizable groups were characterized primarily in terms of accessible surface area, depth of burial, polarity and hydrophobicity of their microenvironments, and contact with other surface or internal ionizable groups and with crystallographic water molecules. MD simulations were also performed on a small set of proteins to examine the robustness of static structures for purposes of determination of depth of burial.