(526f) Molecular Determinants for Protein Stabilization By Insertional Fusion to a Thermophilic Host Protein Domain

Protein stability is a fundamental problem, which may determine the scope of protein applications. While many conventional stabilization methods have garnered some success, they usually cause unwanted side effects, such as compromise in intrinsic biological activity. Moreover, many of these methods lack generality and are not efficient for stabilization of proteins inactivating through various mechanisms, such as reversible and irreversible thermal unfolding with and without formation of aggregates. Therefore the scope of the conventional stabilization methods is highly limited and heavily target protein-dependent. Improved stability of proteins can also promote their evolvability through facilitated extensive sequence variation. Unfortunately, mutation-mediated conventional protein stabilization may require inclusion or exclusion of specific residues in selected locations and thus restrict sequences spaces for additional functional evolution.

Previously, our lab developed a new method to stabilize a target unstable protein through its insertional fusion to a hyperthermophilic maltodextrin binding protein from Pyrococcus furiosus (PfMBP). One clear advantage of our approach stems from the fact that stabilization primarily derives not from changes in the amino acid sequence of the target protein, but from the insertion into a protein chaperone (i.e., PfMBP). Thus, the method is potentially very general and accomplishes stabilization with little, if any, negative effect on the desired properties of the target proteins (e.g., their enzymatic activities and expression levels). Despite the promising observations, the major determinants for stabilization by insertional fusion into PfMBP have yet to be thoroughly examined.

In the presented study, we examined the molecular determinants of protein stabilization by insertion into PfMBP through construction and characterization of a set of insertional fusion complexes using various β-lactamase (BLA) homologues as well as a BLA backbone-cyclized variant. We found that insertional fusion to PfMBP affected the relative energetic states of folded, unfolded and intermediate forms of BLA by enthalpic and entropic means, which were associated with inter-domain interactions and reduced terminal entropy of the guest protein domain, respectively. Importantly, the implication of the two different, complementary stabilizing means working in different inactivation pathways significantly increases general applicability of our stabilization method. Insertional fusion to PfMBP also significantly decreased aggregation of the BLA domain and increased its kinetic stability at high temperatures. Thus, enhanced stabilization by insertion may serve for facilitating laboratory evolution of proteins without limiting sequence spaces to explore. Collectively, our thermodynamic and kinetic analyses using comprehensive biochemical/biophysical tools have shed light into an understanding of how insertion into PfMBP stabilized proteins.