Abstract
Engineered proteins possess nearly limitless possibilities in medical and industrial applications but finding a precise amino acid sequence for these applications is challenging. A robust approach for discovering protein sequences with a desired functionality uses a library design method in which combinations of mutations are applied to a robust starting point. Determining useful mutations can be tortuous, yet rewarding; in this chapter, we present a novel library design method that uses information provided by ancestral sequence reconstruction (ASR) to create a library likely to have stable proteins with diverse function. ASR computational tools use a multi-sequence alignment of homologous proteins and an evolutionary model to estimate the protein sequences of the numerous common ancestors. For all ancestors, these tools calculate the probability of every amino acid occurring at each position within the sequence alignment. The alternate amino acid states at individual positions corelate to a region of stability in sequence space around the ancestral sequence which can inform site-wise diversification within a combinatorial library. The method presented in this chapter balances the quality of results, the computational resources needed, and ease of use.
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References
Siegel JB, Zanghellini A, Lovick HM et al (2010) Computational design of an enzyme catalyst for a stereoselective bimolecular diels-alder reaction. Science 329:309–313
Hyster TK, Knörr L, Ward TR et al (2012) Biotinylated Rh(III) complexes in engineered streptavidin for accelerated asymmetric C-H activation. Science 338:500–503
Coelho PS, Brustad EM, Kannan A et al (2013) Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339:307–310
Chen F, Gaucher EA, Leal NA et al. (2010) Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection. Proceedings of the National Academy of Sciences of the United States of America, 107(5), 1948–1953. https://doi.org/10.1073/pnas.0908463107
Gaucher EA, Govindarajan S, Ganesh OK (2008) Palaeotemperature trend for Precambrian life inferred from resurrected proteins. Nature 451:704–707
Risso VA, Gavira JA, Sanchez-Ruiz JM (2014) Thermostable and promiscuous Precambrian proteins. Environ Microbiol 16:1485–1489
Nguyen V, Wilson C, Hoemberger M et al (2017) Evolutionary drivers of thermoadaptation in enzyme catalysis. Science 355:289–294
Bloom JD, Labthavikul ST, Otey CR et al (2006) Protein stability promotes evolvability. Proc Natl Acad Sci U S A 103:5869–5874
Risso VA, Gavira JA, Mejia-Carmona DF et al (2013) Hyperstability and substrate promiscuity in laboratory resurrections of precambrian β-lactamases. J Am Chem Soc 135:2899–2902
Thornton JW, Need E, Crews D (2003) Resurrecting the ancestral steroid receptor: ancient origin of estrogen signaling. Science 301:1714–1717
Bar-Rogovsky H, Stern A, Penn O et al (2015) Assessing the prediction fidelity of ancestral reconstruction by a library approach. Protein Eng Des Sel 28:507–518
Eick GN, Bridgham JT, Anderson DP et al (2017) Robustness of reconstructed ancestral protein functions to statistical uncertainty. Mol Biol Evol 34:247–261
Wheeler LC, Harms MJ (2021) Were ancestral proteins less specific? Mol Biol Evol 38(6):1–35
Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797
Nguyen L-T, Schmidt HA, von Haeseler A et al (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274
Minh BQ, Schmidt HA, Chernomor O et al (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37:1530–1534
Jacobs TM, Yumerefendi H, Kuhlman B et al (2015) SwiftLib: rapid degenerate-codon-library optimization through dynamic programming. Nucleic Acids Res 43:1–9
Minh BQ, Nguyen MAT, von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 30:1188–1195
Hoang DT, Chernomor O, von Haeseler A et al (2018) UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35:518–522
Guindon S, Dufayard JF, Lefort V et al (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321
Woldring DR, Holec PV, Zhou H et al (2015) High-throughput ligand discovery reveals a sitewise gradient of diversity in broadly evolved hydrophilic fibronectin domains. PLoS One 10:e0138956
Woldring DR, Holec PV, Stern LA et al (2017) A gradient of sitewise diversity promotes evolutionary fitness for binder discovery in a three-helix bundle protein scaffold. Biochemistry 56:1656–1671
Kruziki MA, Bhatnagar S, Woldring DR et al (2015) A 45-amino-acid scaffold mined from the PDB for high-affinity ligand engineering. Chem Biol 22:946–956
Kruziki MA, Sarma V, Hackel BJ (2018) Constrained combinatorial libraries of Gp2 proteins enhance discovery of PD-L1 binders. ACS Comb Sci 20:423–435
Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques 48:463–465
Schimming O, Fleischhacker F, Nollmann FI et al (2014) Yeast homologous recombination cloning leading to the novel peptides ambactin and xenolindicin. Chembiochem 15:1290–1294
An Y, Ji J, Wu W et al (2005) A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR. Appl Microbiol Biotechnol 68:774–778
Benatuil L, Perez JM, Belk J et al (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159
Khan RT, Musil M, Stourac J et al (2021) Fully automated ancestral sequence reconstruction using FireProtASR. Curr Protoc 1:1–13
Suchard MA, Redelings BD (2006) BAli-Phy: simultaneous Bayesian inference of alignment and phylogeny. Bioinformatics 22:2047–2048
Williams PD, Pollock DD, Blackburne BP et al (2006) Assessing the accuracy of ancestral protein reconstruction methods. PLoS Comput Biol 2:0598–0605
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Thomas A, Cutlan R, Finnigan W et al (2019) Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction. Commun Biol 2:429
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VanAntwerp, J., Finneran, P., Dolgikh, B., Woldring, D. (2022). Ancestral Sequence Reconstruction and Alternate Amino Acid States Guide Protein Library Design for Directed Evolution. In: Traxlmayr, M.W. (eds) Yeast Surface Display. Methods in Molecular Biology, vol 2491. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2285-8_4
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DOI: https://doi.org/10.1007/978-1-0716-2285-8_4
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