Dissecting Amino Acid Osmolytes: Is a Zwitterion Required for Increasing Protein Thermal Stability?

Amino acids are an important class of osmolytes that demonstrate the ability to stabilize certain proteins under acute thermal stress. However, little consensus exists on what structural aspects produce this phenomenon. It has been proposed that the stabilizing nature of amino acids emerges from their zwitterionic nature at neutral pH, but little evidence has supported this view, especially in regards to the breadth of its applicability to proteins of varying structure and composition. In this report, we structurally dissect the amino acids L‐alanine and β‐alanine to determine functional group contributions to protein‐stabilizing activity on two structurally dissimilar proteins: human C‐reactive protein (CRP) and human myoglobin. The stabilizing effects of the osmolytes (and their structural derivatives) were quantified by Differential Scanning Fluorimetry (DSF) in three metrics: protein melting temperature (Tm), standard free energy change (ΔG o ), and “m”‐values. Our results indicate that the zwitterionic theory holds for myoglobin, as removal of either the carboxylate or amino moiety from the amino acid abrogated all thermal stabilizing effects. Furthermore, substitution of the carboxylate group with a hydroxyl group did not restore protein‐stabilizing activity, arguing for the zwitterionic requirement for amino acid‐mediated thermal stabilization. However, the results with CRP were significantly different from those observed with myoglobin. In the case of CRP, the amino acid carboxylate proved to be the dominant stabilizing group, as compounds lacking the amino group conferred greater thermal stability than the complete amino acids. These results affirm that the zwitterionic theory is limited in its ability to describe amino acid‐mediated effects on protein thermal stability and suggests that the nature of the interactions between amino acid osmolytes and proteins is highly dependent on protein structure. These results may have significant implications for the development of chemistries to stabilize diverse protein targets in biospecimens for diagnostic applications. Support or Funding Information California State University Program for Education and Research in Biotechnology (CSUPERB) Chevron Biotechnology Applied Research Endowment Bill and Linda Frost Fund