Suppression of Thermal Protein Aggregation by Acetylation of Lysine Side Chains.

The association of proteins into amorphous aggregates is linked to various detrimental phenomena, ranging from disease mechanisms to the degradation of biopharmaceuticals. The development of aggregation suppression strategies is of considerable interest. Experiments on aggregation (and aggregation suppression) are commonly performed under accelerated conditions, where proteins are exposed to elevated temperatures. Focusing on the thermal aggregation of myoglobin (Mb, a paradigmatic model protein), our group previously reported that intermolecular Lys+/Glu- and Lys+/Asp- salt bridges are a key driver of aggregate formation. Additives such as free arginine and guanidinium salts suppress aggregation by "blocking" Glu- and Asp- side chains, thereby preventing them from participating in salt bridges (Ng & Konermann (2024) JACS 146, 8394). However, the use of such additives, e.g., as excipients in biopharmaceutical formulations, can be problematic. The current work highlights a complementary approach. Using spectroscopic experiments and molecular dynamics simulations, we show that acetylation of Lys side chains is highly effective for suppressing the thermal aggregation of Mb. Acetylation (R-NH3+ → R-NH-CO-CH3) eliminates the positive charge of Lys, thereby rendering the corresponding side chains incapable of salt bridge formation. It is demonstrated that acetylation only has relatively minor effects on the native protein structure under physiological conditions (room temperature, pH 7). In summary, Lys acetylation is a simple yet effective aggregation suppression approach that may also be applicable to other proteins, including biopharmaceuticals such as monoclonal antibodies.