Abstract
Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.
Highlights
Polypeptides fold and adopt three dimentional structures suitable for specific functions
Zn was incorporated for ordering of tertiary structure and organization of the catalytic apparatus by coordination to a triad of His residues in a molecular pocket of the abb fold
The catalytic Zn complex was chosen to be at the base of a funnelshaped pocket and side chains suitable for substrate binding were placed on the rim of the funnel
Summary
Polypeptides fold and adopt three dimentional structures suitable for specific functions. The physical basis of protein folding as a function of amino acid sequence remains unclear.[1] One approach to understanding the basis for folding is protein design, which tests our knowledge of molecular determinants of structure, stability and function. Artificial proteins have since been created, and natural proteins have been retrofitted to possess desired functions as sensors and enzymes.[8,9,10,11,12] These successes probably reflect the success of protein evolution by optimizing sequences to introduce novel activity on pre-existing folds. The independence of evolution of function from evolution of fold-structure suggests a design approach in which first folds-structure and sequences over the folds may be designed to incorporate specific activity independently
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