Are Proteins Charged?

Proteins are charged due to the presence of ionizable residues that can release or take up protons, leading to distinct charge microstates. The extent of protonation is influenced by the overall pH of the solution and the local environments of ionizable residues. The overall partition function is a sum over all charge microstates and Boltzmann weights of all conformations associated with each microstate. The charge microstates can be grouped into mesostates, where each mesostate is a collection of microstates of the same net charge. The pKa values of ionizable residues and the relative mesostate populations as a function of pH can be estimated using the q-canonical ensemble approach, which allows for the decoupling of contributions of net proton binding from those of conformational equilibria.

Charge State Heterogeneity of Proteins

The charge state heterogeneity of proteins with multiple ionizable residues can be quantified using potentiometric measurements of net charge and the q-canonical ensemble approach. This method provides protein-specific quantitative descriptions of pH-dependent populations of mesostates, which is of direct relevance for measuring and understanding how different charge states contribute to conformational, binding, and phase equilibria of proteins.

Ionizable Residues in Proteins

Proteins contain various ionizable amino acid residues, such as:

1. Acidic Residues: Aspartic acid (Asp) and Glutamic acid (Glu) have carboxyl groups (-COOH) that can lose a proton (H+) and become negatively charged (-COO-).
2. Basic Residues: Lysine (Lys), Arginine (Arg), and Histidine (His) have amino groups (-NH2) that can gain a proton (H+) and become positively charged (-NH3+).
3. Neutral Residues: Serine (Ser), Threonine (Thr), Tyrosine (Tyr), and Cysteine (Cys) have hydroxyl groups (-OH) that can also be ionized, depending on the pH.

The ionization state of these residues is determined by the pH of the surrounding environment and the specific pKa values of the ionizable groups. The pKa values can be influenced by the local environment, such as the presence of other charged groups, hydrogen bonding, and the overall protein structure.

Quantifying Charge State Heterogeneity

The charge state heterogeneity of proteins can be quantified using potentiometric measurements of net charge and the q-canonical ensemble approach. This method allows for the determination of the relative populations of different charge states (mesostates) as a function of pH.

The key steps in this approach are:

1. Potentiometric Titration: Measuring the net charge of the protein as a function of pH using potentiometric titration experiments.
2. q-Canonical Ensemble: Applying the q-canonical ensemble approach to model the pH-dependent populations of different charge states (mesostates) based on the pKa values of the ionizable residues and the protein’s conformational equilibria.
3. Mesostate Populations: Obtaining protein-specific quantitative descriptions of the pH-dependent populations of different charge state mesostates, which are relevant for understanding the contributions of various charge states to conformational, binding, and phase equilibria of the protein.

This approach provides a comprehensive understanding of the charge state heterogeneity of proteins, which is crucial for studying their structure, function, and interactions in various biological processes.

Proteins in the Intracellular Environment

In living cells, proteins function in a heterogeneous and crowded intracellular environment, where the properties of proteins and nucleic acids can be significantly altered compared to buffer alone. The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed, leading to a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions.

Measuring Protein Stability in Living Cells

NMR-detected hydrogen–deuterium exchange (HDX) of quenched cell lysates can be used to measure individual opening free energies of proteins in living cells without adding destabilizing cosolutes or heat. This approach provides a more complete understanding of the effects of the intracellular environment on protein chemistry, including the impact on protein charge states and their contributions to protein stability, folding, and function.

The key steps in this HDX-based approach are:

1. Cell Lysis: Quenching the cells to preserve the native state of proteins and prevent further changes in the intracellular environment.
2. Hydrogen-Deuterium Exchange: Exposing the quenched cell lysates to deuterium-containing buffer, allowing for the exchange of hydrogen atoms with deuterium atoms in the protein backbone.
3. NMR Analysis: Using NMR spectroscopy to detect the extent of hydrogen-deuterium exchange at the residue level, providing information about the opening free energies of individual structural elements within the protein.

This method allows for the measurement of protein stability and dynamics in living cells, without the need for adding destabilizing cosolutes or heat, which can alter the native properties of the proteins.

Conclusion

In summary, proteins are charged due to the presence of ionizable residues, and the charge state heterogeneity of proteins can be quantified using potentiometric measurements and the q-canonical ensemble approach. The properties of proteins in living cells can be significantly altered compared to buffer alone, and NMR-detected hydrogen–deuterium exchange of quenched cell lysates can be used to measure individual opening free energies of proteins in living cells without adding destabilizing cosolutes or heat, providing a more complete understanding of the effects of the intracellular environment on protein chemistry.

References:

1. Fossat, M. J., Posey, A. E., & Pappu, R. V. (2021). Quantifying charge state heterogeneity for proteins with multiple ionizable residues. Biophysical Journal, 120(23), 5089-5101. https://www.sciencedirect.com/science/article/pii/S0006349521038613
2. Rosen, J., Forman, S. A., Agarwal, V., & Gruebele, M. (2014). Residue level quantification of protein stability in living cells. Proceedings of the National Academy of Sciences, 111(33), 11929-11934. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128145/
3. Gitlin, I., Carbeck, J. D., & Whitesides, G. M. (2006). Why are proteins charged? Networks of charge-charge interactions in proteins measured by charge ladders and capillary electrophoresis. Angewandte Chemie International Edition, 45(19), 3022-3060. https://www.researchgate.net/publication/7161562_Why_Are_Proteins_Charged_Networks_of_Charge-Charge_Interactions_in_Proteins_Measured_by_Charge_Ladders_and_Capillary_Electrophoresis
4. Gitlin, I., Carbeck, J. D., & Whitesides, G. M. (2006). Why are proteins charged? Networks of charge–charge interactions in proteins measured by charge ladders and capillary electrophoresis. Angewandte Chemie International Edition, 45(19), 3022-3060. https://onlinelibrary.wiley.com/doi/10.1002/anie.200502530