Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization

Enhancing the thermostability
of enzymes without impacting their
catalytic function represents an important yet challenging goal in
protein engineering and biocatalysis. We recently introduced a novel
method for enzyme thermostabilization that relies on the computationally
guided installation of genetically encoded thioether “staples”
into a protein via cysteine alkylation with the noncanonical amino
acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate
the functionality of an expanded set of electrophilic amino acids
featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain
groups for protein stapling using this strategy. Using a myoglobin-based
cyclopropanase as a model enzyme, our studies show that covalent stapling
with p-chloroacetamido-phenylalanine (pCaaF) provides
higher stapling efficiency and enhanced stability (thermodynamic and
kinetic) compared to the other stapled variants and the parent protein.
Interestingly, molecular simulations of conformational flexibility
of the cross-links show that the pCaaF staple allows fewer energetically
feasible conformers than the other staples, and this property may
be a broader indicator of stability enhancement. Using this strategy,
pCaaF-stapled variants with significantly enhanced stability against
thermal denaturation (ΔTm′
= +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high
levels of catalytic activity and stereoselectivity. Crystallographic
analyses of singly and doubly stapled variants provide key insights
into the structural basis for stabilization, which includes both direct
interactions of the staples with protein residues and indirect interactions
through adjacent residues involved in heme binding. This work expands
the toolbox of protein stapling strategies available for protein stabilization.