Evolutionary paths that link orthogonal pairs of binding proteins.

Some protein binding pairs exhibit extreme specificities that functionally insulate them from homologs. Such pairs evolve mostly by accumulating single-point mutations, and mutants are selected if their affinity exceeds the threshold required for function. Thus, homologous and high-specificity binding pairs bring to light an evolutionary conundrum: how does a new specificity evolve while maintaining the required affinity in each intermediate? Until now, a fully functional single-mutation path that connects two orthogonal pairs has only been described where the pairs were mutationally close thus enabling experimental enumeration of all intermediates.

Design and in vitro realization of carbon-conserving photorespiration.

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss.

Automated Design of Efficient and Functionally Diverse Enzyme Repertoires.

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations.