Designed active-site library reveals thousands of functional GFP variants.

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.

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 function1-4. 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 intermediate5,6? 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 intermediates2. We present an atomistic and graph-theoretical framework for discovering low molecular strain single-mutation paths that connect two extant pairs, enabling enumeration beyond experimental capability. We apply it to two orthogonal bacterial colicin endonuclease-immunity pairs separated by 17 interface mutations7. We were not able to find a strain-free and functional path in the sequence space defined by the two extant pairs. But including mutations that bridge amino acids that cannot be exchanged through single-nucleotide mutations led us to a strain-free 19-mutation trajectory that is completely viable in vivo. Our experiments show that the specificity switch is remarkably abrupt, resulting from only one radical mutation on each partner. Furthermore, each of the critical specificity-switch mutations increases fitness, demonstrating that functional divergence could be driven by positive Darwinian selection. These results reveal how even radical functional changes in an epistatic fitness landscape may evolve.