Published on Wed Sep 01 2021

Quantifying charge state heterogeneity for proteins with multiple ionizable residues

Fossat, M. J., Posey, A. E., Pappu, R. V.

For proteins with multiple ionizable residues, the canonical assumption is that ionization states of residues are fixed by their intrinsic pKa values. We analyze data for global charge vs. pH to extract mesostate populations as a function of pH. Our findings reveal that the heterogeneity of charge states makes significant contributions to measured charge profiles.

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Abstract

For proteins with multiple ionizable residues, the canonical assumption is that ionization states of residues are fixed by their intrinsic pKa values. However, several studies have shown that protonation / deprotonation of acidic vs. basic sidechains is realizable even when the solution pH is kept fixed at values that are far away from the intrinsic pKa values. Indeed, protein solutions are best described as ensembles of charge microstates, with each member of the ensemble being a distinct charge microstate defined by differences in charge states for ionizable residues. Accordingly, for a given set of solution conditions, the true partition function is sum over all charge microstates and all the Boltzmann weights of all conformations associated with each of the charge microstates. Here, we leverage the advantages afforded by potentiometric titrations to measure global net charge as a function of pH, independent of considerations of conformational preferences. The systems studied are fragments of proteins with repetitive patterns of Lys and Glu. We analyze the potentiometry data using the recently introduced formalism of the q-canonical ensemble. In this ensemble, charge microstates can be grouped into mesostates. Each mesostate is a collection of microstates of the same net charge. We analyze data for global charge vs. pH to extract mesostate populations as a function of pH. Our findings reveal that the heterogeneity of charge states makes significant contributions to measured charge profiles. This has significant implications for the types of species that are present in solution, even for a fixed pH. Measurements of net charge, decoupled from measurements of conformational equilibria, and analyzed to extract the pH-dependent populations of different mesostates, will be significant for accurate understanding of how charge state heterogeneity contributes to conformational, binding, and phase equilibria of proteins, especially those that are intrinsically disordered.