Connecting high-temperature and low-temperature protein stability and aggregation

Abstract

Protein aggregation is a long-standing problem for preservation of proteins in both laboratory settings and for commercial biotechnology products. It is well established that heating (cooling) can accelerate (slow) aggregation by populating (depopulating) unfolded or partially unfolded monomer states that are key intermediates in aggregation processes. However, there is a long-standing question of whether the same mechanism(s) that lead to aggregation under high-temperature stress are relevant for low-temperature stress such as in refrigerated or supercooled liquids. This report shows the first direct comparison of “hot” and “cold” aggregation kinetics and folding/unfolding thermodynamics, using bovine hemoglobin as a model system. The results suggest that the same mechanism for non-native aggregation holds from “hot” to “cold” temperatures, with an aggregation temperature-of-maximum-stability slightly below 0°C. This highlights that sub-zero temperatures can induce cold-mediated aggregation, even in the absence of freezing stresses. From a practical perspective, the results suggests the possibility that cold-stress may be a useful alternative to heat-stress for extrapolating predictions of protein shelf life at refrigerated conditions, as well as providing a foundation for more mechanistic studies of cold-stress conditions in future work. A comparison between isochoric and isobaric methods is also briefly discussed.

Thermodynamic phase diagram of amyloid-β (16–22) peptide

“…This suggests that our computation-based approach could inform future fabrication of amyloid nanofiber substrates/scaffolds with desired microstructure properties for various tissue engineering and biomedical applications (47,48). In addition, controlling protein aggregation is a key problem for the purification and storage of therapeutic proteins (49,50). Thus, knowledge of the solubility of specific peptides and proteins over a wide range of temperatures could be used to effectively prevent their unwanted agglomeration.…”

Hyperthermostable cube-shaped assembly in water

“…Although Eq. (1) is a theoretical equation that is valid only when ΔC P is constant with temperature, the destabilisation of proteins at decreasing temperature was truly observed 39,40 . Figure 6a, b…”

The Molecular Interaction Process

“…Currently a debate, similar in nature to the polymer/colloid one, is occurring today: is aggregation governed by molecular (sometimes focused on conformational) properties or colloidal (sometimes referred to as physical) properties? 12,29,30,32,33,35 Figure 1 presents one way to consider this question. In it, the molecular properties are divided into 3 categories: native-state variants (or native-like structures), conformational variants, and chemical variants, which encompasses any covalent structural variants (glycosylations, oxidations, deamidations, etc.).…”

“…6,7,[10][11][12][13][24][25][26][27][28] In many cases, a goal of the research has been to link the molecular properties of proteins, such as conformational stability, charge, dipole moment, charge fluctuation, solvation, hydrophobicity, and protein flexibility to these solution properties. [12][13][14][15][16][29][30][31][32][33][34][35][36] For monoclonal antibody aggregation there is ongoing discussions of which domain(s) are responsible for the aggregation and whether the aggregation proceeds through native or non-native structures. 6,7,24,30,34,[36][37][38][39] While these are important and detailed questions, it is worthwhile to step back and consider the common processes and energetics that underlie molecular interactions.…”