Peptides containing amino acids with ionisable side chains represent a typical example of weak ampholytes, that is, molecules with multiple titratable acid and base groups, which generally exhibit charge regulating properties upon changes in pH. Charged groups on an ampholyte interact electrostatically with each other, and their interaction is coupled to conformation of the (macro)molecule, resulting in a complex feedback loop. Their charge-regulating properties are primarily determined by the pKA of individual ionisable side-chains, modulated by electrostatic interactions between the charged groups. The latter is determined by the amino acid sequence in the peptide chain. In our previous work we introduced a simple coarse-grained model of a flexible peptide. We validated it against experiments, demonstrating its ability to quantitatively predict charge on various peptides in a broad range of pH. In the current work, we investigated two types of peptide sequences: diblock and alternating, each of them consisting of an equal number of amino acids with acid and base side-chains. We showed that changing the sequence while keeping the same overall composition has a profound effect on the conformation, whereas it practically does not affect total charge on the peptide. Nevertheless, the sequence significantly affects the charge state of individual groups, showing that the zero net effect on the total charge is a consequence of unexpected cancellation of effects. Furthermore, we investigated how the difference between the pKA of acid and base side chains affects the charge and conformation of the peptide, showing that it is possible to tune the charge-regulating properties by following simple guiding principles based on the pKA and on the amino acid sequence. Our current results provide a theoretical basis for understanding of the complex coupling between the ionisation and conformation in flexible polyampholytes, including synthetic polymers, biomimetic materials and biological molecules, such as intrinsically disordered proteins, whose function can be regulated by changes in the pH.

Weak ampholytes are ubiquitous in nature and commonly found in artificial pH-responsive systems. However, our limited understanding of their charge regulation and the lack of predictive capabilities hinder the bottom-up...

In recent experiments, the “local pH” near polyelectrolyte chains was determined from the shift in the effective acidity constant of fluorescent pH indicators attached to the macromolecules. This indirect determination raises the question if the analyzed quantity was indeed the “local pH” and what this term actually means. In this study, we combined experiments and simulations to demonstrate that the shift in ionization constant is slightly lower than the difference between the pH and the “local pH”. This offset is caused by correlations between fluctuations in chain conformation, small-ion distribution, and fluorophore ionization.

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