Quantifying the Effects of Charge Regulation on Disorder-Order Transitions of Highly Charged Protein Sequences

Abstract

Charge regulation, which refers to the modulation of charge states of ionizable residues due to shifted pKa values, is an important albeit often overlooked determinant of sequence-ensemble and sequence-structure relationships of intrinsically disordered and intrinsically ordered proteins. We have developed complimentary computational and experimental approaches that account for the complete ensemble of charge states for a given sequence. This combined approach enables the computational calculation of conformational ensembles as a function of pH, and experimental measurement of charge states as a function of pH, allowing us to quantify the effects of charge regulation on archetypal sequences that are predicted to be intrinsically disordered. Here, we present results from a joint experimental and computational analysis of the effects of charge regulation on block-copolypeptides composed entirely of acidic and basic residues, (E4K4)n. These sequences form stable single alpha helices, although they are erroneously predicted to form disordered ensembles if one assumes fixed charge states for the ionizable residues. We employ a novel capillary electrophoresis method for measuring charge as a function of pH. These data, combined with potentiometric analysis, show that glutamate residues have highly shifted pKa values when compared to those of model compounds. This preferential neutralization of glutamate residues that are internal to the block co-polypeptide sequence engenders alpha helix formation by lowering the free energy of desolvation and enabling the favorable propagation of the alpha helix. These findings are readily explained using simulations based on the recently developed q-canonical Monte Carlo sampling method. Our results suggest that sequence-specific charge regulation can engender large-scale conformational transitions and even underlie the driving forces for phase separation. Further, our results highlight the need for refining disorder predictors based on an improved understanding of context dependent charge regulation effects.