Site-selective control of the reactivity of surface-exposed histidine residues in designed four-helix-bundle catalysts

Background: The structure and function of native proteins often depend on the interplay between ionisable residues with physical properties that have been fine tuned by interactions with neighbouring groups. Here, we systematically vary the environment of histidines in designed helix-loop-helix motifs to modulate histidine pK(a) values and reactivities. Results: 25 helix-loop-helix motifs were designed in which surface-exposed histidine residues were flanked by neutral, negatively charged and positively charged groups and the histidine's proximity to the hydrophobic core was varied. The 57 histidine pK(a) values were determined by H-1 NMR spectroscopy and found to be in the interval 5.2-7.2 with changes ranging from a decrease of 1.3 pK(a) units to an increase of 0.7 pK(a) units compared with the pK(a) for an unperturbed histidine residue. Conclusions: A decrease in the pK(a) of His34 by 1.3 units was accomplished by placing it in close proximity to the hydrophobic core and flanking it by positively charged residues in positions (i, i + 3) and (i, i - 4). Flanking a histidine residue with a lysine or a histidine in positions (i, i + 3), (i, i + 4) or (i, i - 4) resulted in pK(a) depressions of similar to 0.5 pK(a) units per residue and additivity was observed. The increase of the histidine pK(a) by glutamate residues was the most efficient in position (i, i + 3), but less efficient in position (i, i + 4). These principles should be useful in the engineering of novel catalysts.