Assessing the prediction fidelity of ancestral reconstruction by a library approach.
Ancestral reconstruction is a powerful tool for studying protein evolution as well as for protein design and engineering. However, in many positions alternative predictions with relatively high marginal probabilities exist, and thus the prediction comprises an ensemble of near-ancestor sequences that relate to the historical ancestor. The ancestral phenotype should therefore be explored for the entire ensemble, rather than for the sequence comprising the most probable amino acid at all positions [the most probable ancestor (mpa)]. To this end, we constructed libraries that sample ensembles of near-ancestor sequences. Specifically, we identified positions where alternatively predicted amino acids are likely to affect the ancestor's structure and/or function. Using the serum paraoxonases (PONs) enzyme family as a test case, we constructed libraries that combinatorially sample these alternatives. We next characterized these libraries, reflecting the vertebrate and mammalian PON ancestors. We found that the mpa of vertebrate PONs represented only one out of many different enzymatic phenotypes displayed by its ensemble. The mammalian ancestral library, however, exhibited a homogeneous phenotype that was well represented by the mpa. Our library design strategy that samples near-ancestor ensembles at potentially critical positions therefore provides a systematic way of examining the robustness of inferred ancestral phenotypes.
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Hagit
H
Bar-Rogovsky
Department of Cell Research and Immunology, George. S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Adi
A
Stern
Department of Cell Research and Immunology, George. S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Osnat
O
Penn
Entelechon GmbH, Industriestraße 1, 93077 Bad Abbach, Germany.
Iris
I
Kobl
Department of Cell Research and Immunology, George. S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Tal
T
Pupko
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel tawfik@weizmann.ac.il.
Dan S
DS
Tawfik
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D006801:Humans; D008322:Mammals / Q000235:genetics; D008958:Models, Molecular; D010641:Phenotype; D010802:Phylogeny; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
D002265:Carboxylic Ester Hydrolases; C019734:arylesterase
ancestral sequence reconstruction; inferred ancestor; predicted ancestor; serum paraoxonase