Impact of charge regulation on conformational and phase equilibria of intrinsically disordered proteins

Cells are organized into distinct compartments, and many of these compartments are defined by different acidity. An environment rich in protons can change the charge on proteins in cells if the proteins have amino acids that are ionizable. Changes to charge states of proteins that arise from the association or dissociation of protons can control how proteins in cells respond to specific types of signals such as environmental factors that induce changes to salt or proton concentrations.

Research in this project will leverage new methods to study how specific proteins, known as intrinsically disordered proteins, are modified by the uptake or release of protons based on the acidity of the solution. Studies carried out as part of the project will lead to an improved understanding of how alterations to charge states of proteins influence their functions.

This work is important because charge-mediated interactions are central to a variety of protein functions including the recognition of one protein by another. Given the unique nature of the technological advances that are driving research in the project, there will be novel training opportunities at the intersection of computation and experiment in the broad area of molecular biophysics.

Findings from the project will be incorporated into materials of courses for undergraduates. Further, key personnel who are involved in the project will work closely with students from inner city middle and high-schools to explain how pH and a better understanding of acid-base equilibria will drive new research frontiers that emerge from the project.

The blend of computation and experiment, driven in part by the development of novel computational tools, including machine learning modules, will create new workflows for solving challenging problems in molecular biophysics. These modules will be incorporated into instructional modules for research trainees and students in molecular biophysics.

The goal of the project is to understand how the exchange of protons between ionizable residues and the accumulation of solution ions around these residues contribute to the charge states and conformations of intrinsically disordered proteins (IDPs). Currently, our understanding of the form, functions, and phase behaviors of IDPs is limited by the simplifying assumption that charge states of ionizable residues are fixed by the pKa values of model compounds.

The proposed work rests on the principle that conformational fluctuations engender changes to local microenvironments of ionizable residues. These local heterogeneities can induce exchange of protons among titratable sites. This can also induce preferential accumulation of solution ions around ionizable residues.

A key advance that drives the investigations is the recent development of the q-canonical ensemble, which is a formal and rigorous statistical physics-based description of the joint effects of charge state and conformational heterogeneity. Importantly, an integrated experimental and computational pipeline has been developed and this allows one to leverage the structure of the q-canonical ensemble to quantify the effects of proton association or dissociation, known as charge regulation, and its interplay with the effects of solution ions through charge renormalization.

Approaches that rest on the formal decoupling between measurements of charge and conformation, will allow for the incorporation of information derived from potentiometric titrations and measurements of conformational or phase equilibria into simulations. This, when anchored by experimental data, will lead to a full description of the ensemble of charge state and conformations that are accessible at different solution conditions.

The approaches are novel, the focus is unique, and the insights to be generated from the project are likely to be unprecedented. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.