Structural Reorganization Triggered by the Ionization of Lys Residues buried in Hydrophobic Environments

Abstract

A study of the structural consequences of ionization of amino acid side chains buried in the hydrophobic interior of proteins was performed by systematic introduction of lysine at 25 internal positions in a highly stable form of staphylococcal nuclease. Crystal structures for 12 of the variants at pH values where the Lys was expected to be uncharged revealed nearly identical backbones with the reference protein and ordered internal Lys side chains at the substitution site. The structures demonstrate that neutral Lys can be buried in the protein interior without the need for any specialized structural adaptations save for those required to stabilize the protein. Solvent-accessible surface area and depth of burial calculations indicated that all but one Lys were completely inaccessible to water. Seven Lys side chains packed into predominantly hydrophobic regions; five had Nζ atoms within 3.5 A of one or more polar groups or crystallographic water molecules. Depth of burial or microenvironment polarity as observed in the crystal structures did not correlate with measured shifts in pKa. 1H-15N HSQC NMR spectroscopy studies revealed global unfolding coupled to the ionization of two of the 25 Lys residues. Three others showed partial or local unfolding, five demonstrated localized changes in structure and dynamics, and 15 exhibited no detectable consequences. Backbone amide resonances were most similar between reference protein and the Lys-containing variant when the Lys side chains were buried shallowly or in a loop region. In the least stable variants the ionization of the internal Lys residues led to localized increase in conformational fluctuations or to conformational reorganization. These data will be useful to test the ability of structure-based pKa calculations to account for structural rearrangements or increased dynamics in response to ionization of internal groups.