De novo enzyme design: a novel Kemp eliminase

Privett, H.K., Kiss, G., Lee, T.M., Blomberg, R., Chica, R.A., Thomas, L.M., Hilvert, D., Houk, K.N. & Mayo, S.L. (2012). Iterative approach to computational enzyme design. Proc Natl Acad Sci USA 109, 3790–3795

Kinetic characterization (Michaelis-Menton plot) showing catalytic activity of designed variants compared to wild-type protein (scaffold only), which is inactive.


Privett et al. combined iterative rounds of computational protein design and structural analyses to design Kemp eliminase activity into a  previously inert protein scaffold.  The resulting protein represents the most catalytically efficient computationally designed enzyme to date, with kcat/kuncat = 1.5 x 106. This design was subsequently used as a starting point for directed evolution, which yielded variants with even further improved activity.

Crystal structures of designed positions in two of the most red-shifted mutants. The chromophore is shown in magenta. H-bonds are indicated by dashed lines.

Improved spectral properties: a longer emission wavelength red fluorescent protein

Chica et al. applied a combined computational and experimental approach that uses computational protein design as an in silico prescreen to generate focused combinatorial libraries of mutant sequences of mCherry, a red fluorescent protein. The computational procedure helped identify residues that could fulfill interactions hypothesized to cause red shifts without destabilizing the protein fold. Screening of the focused library resulted in three mutants with emission wavelengths that were red-shifted 20–26 nm.

This approach was very efficient as it required the experimental screening of a total of ∼5,000 clones, several orders of magnitude less than what has been required to achieve comparable red shifts using directed evolution alone. Crystal structures of two of the mutants confirmed the interactions hypothesized to cause red shifts.

X-ray structure of CaM (blue) in complex with one of its targets CaMKIIp (PDB code: 1CM1) (red). CaM residues included in the optimization of the CaM–CaMKIIp binding interface are shown in green and calcium ions are shown as yellow spheres.

Enhanced binding specificity: calmodulin

Yosef et al. used computational protein design to alter calmodulin (CaM) binding specificity to CaM-dependent protein kinase II (CaMKII) and calcineurin. The best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, which is the highest specificity switch achieved in any protein-protein interface through the computational protein design approach.