Designed active-site library reveals thousands of functional GFP variants

Weinstein, J. Y.; Aldaravi, C. M. G.; Lipsh-Sokolik, R.; Hoch, S. Y.; Liebermann, D.; Nevo, R.; Weissman, H.; Petrovich-Kopitman, E.; Margulies, D.; Ivankov, D.; McCandlish, D.; Fleishman, S. J.

doi:10.1101/2022.10.11.511732

bioRxiv · 10.1101/2022.10.11.511732

Designed active-site library reveals thousands of functional GFP variants

Weinstein, J. Y.·Aldaravi, C. M. G.·Lipsh-Sokolik, R.·Hoch, S. Y.·Liebermann, D.·Nevo, R.·Weissman, H.·Petrovich-Kopitman, E.·Margulies, D.·Ivankov, D.·McCandlish, D.·Fleishman, S. J.

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Abstract

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is extremely sensitive to mutations due to a high density of molecular interactions, drastically reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called htFuncLib, to design a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We applied htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recovered >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 {degrees}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 useful for one-shot optimization of activity in enzymes, binders, and other proteins.

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Weinstein, J. Y., Aldaravi, C. M. G., Lipsh-Sokolik, R., Hoch, S. Y., Liebermann, D., Nevo, R., Weissman, H., Petrovich-Kopitman, E., Margulies, D., Ivankov, D., McCandlish, D., Fleishman, S. J.. 2022-10-11. Designed active-site library reveals thousands of functional GFP variants. https://doi.org/10.1101/2022.10.11.511732

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