Aggregation describes the formation of large clusters of proteins. Aggregated proteins often lose their functionality. This can lead to ambiguous results in biochemical and biophysical assays, and aggregates can cause adverse effects when present in therapeutic drugs. Aggregation therefore needs to be avoided for many applications, but it is a common problem during purification, refolding, shipping, and storage, to name some examples. The aggregation behavior of a protein is often investigated as one aspect of protein stability.

Aggregation from the denatured state

Aggregation typically occurs as a consequence of denatured or partially denatured proteins (see illustration). When proteins (partially) unfold, hydrophobic patches from the interior of the protein often become exposed to the surface. In a polar environment (e.g. a buffer), these hydrophobic patches can then interact with hydrophobic patches of other molecules to form aggregates. In more general terms, unfolded regions will probe the conformational space to identify the lowest possible energy state, which is not necessarily the native conformation. Aggregation is a way to minimize the energy level by favoring a maximum of electrostatic and hydrophobic interactions allowed by the amino acid sequence. Partial unfolding can be caused by elevated temperature or by the presence of destabilizing agents, such as DMSO. While (partial) unfolding is potentially reversible, aggregation is typically not. This means that an aggregated protein sample typically cannot be recovered, which makes it important to prevent aggregation from happening in the first place. It also means that strictly speaking, there is no equilibrium in thermal unfolding experiments, because the (irreversible) aggregation process keeps removing unfolded protein from the reaction.

Aggregation from the denatured state is easy to screen for with Prometheus. By screening for conditions that stabilize the native state, which can be done by screening for thermal stability.

Aggregation from the native state

Some proteins, e.g. membrane proteins with large surface-exposed hydrophobic regions or intrinsically disordered proteins, show a tendency to form aggregates even in their native (folded) state. In such cases, solubilizing additives, such as detergents are used to keep these molecules in solution. Aggregation from the native state is best investigated using light-scattering methods.

Figure: This illustration shows different aggregation pathways. Aggregation may originate from the denatured or native state of a protein. (Partially) unfolded molecules start interacting, forming large aggregates. This process can be accelerated by increasing temperature.

Aggregation studies on Prometheus

Prometheus Panta measures protein aggregation via scattering using the DLS optics and turbidity using the backreflection optics. Prometheus Panta outputs the onsets of aggregation (see image below), which are temperatures at which the sample starts to aggregate or changes in size. The different states of aggregation may be reflected in the different onsets, from oligomerization to precipitation. All of these parameters are useful for comparing protein colloidal stability, and are generally useful to know as temperatures to keep your sample below. The onsets of aggregation often correlate with the unfolding of the protein as shown in the figure below. For proteins showing more than one unfolding event, aggregation may happen as a consequence of the first unfolding event or of a later one. Additional information on the underlying pathway of aggregation can be obtained by comparing the scattering, turbidity, and unfolding profiles with each other.