Insufficient stability of enzymes limits their practical applications. While various strategies that improve enzyme stability have been reported, they frequently suffer from a compromise of other intrinsic properties, such as enzymatic activity. Moreover, conventional methods for enzyme stabilization have a limited generality - a method working for one enzyme does not necessarily stabilize other enzymes. Recently, our lab has proposed insertional fusion of an enzyme into a thermophilic maltose binding protein as a potentially general method of thermally stabilizing various enzymes without compromising other intrinsic properties. In the presented study, we examined the molecular determinants of thermal stabilization of beta-lactamase (BLA) upon insertion into the thermophilic maltose binding protein from Pyrococcus furiosus (PfMBP) through construction and characterization of a set of insertional fusion complexes using various BLA homologues. We also characterized thermal stability of insertional fusion complexes differing in spatial orientation and interdomain distance of the two fused domains as well as a backbone-cyclized BLA 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. Insertional fusion to PfMBP also significantly decreased aggregation of the BLA domain and increased its kinetic stability at high temperatures. Collectively, our thermodynamic and kinetic analyses using comprehensive biochemical/biophysical tools have shed light into an understanding of how insertion into PfMBP stabilized BLA.

Molecular determinants for enzyme stabilization by insertional fusion to a thermophilic maltose binding protein

247th National Meeting (2014)

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