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Schlagwörter:
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Zusammenfassung:
Amino acids within a protein sequence interact to maintain their structure and function.
Consequently, mutations at a given functionally signicant position can be potentially com-
pensated by a mutation at an interacting position. For instance, a substitution from an amino
acid with a small side chain to an amino-acid with a big side chain can be compensated by a
reciprocal substitution at an interacting position. Such a coevolution scenario implies that the
rst mutation leads to a tness reduction, while the compensating mutation restores it. Several
methods have been developed to detect coevolving positions from sequence alignments. How-
ever, the validation of the resulting predictions relies so far only on indirect evidence such as
residue contact maps in proteins for which an experimental structure is available. This study
used the dataset that mapped substitutions for a protein sequence alignment on each phylogeny
branch to detect coevolving amino-acids in bacterial homologous protein families (CoMap).
Accounting for the biochemical properties of amino acids, we short-listed potential candidates
from a dataset of thousands of coevolving groups for the experimental assessment. We then
selected three candidate groups from three proteins (Elongation factor 4, IspH, and YebC pro-
teins) displaying a pattern of co-substitutions in the Escherichia coli branch of the phylogeny for
charge and beta propensities for compensation. I experimentally reconstructed the local tness
landscape, resurrecting the ancestral genotype in E. coli and putting it in competition with sin-
gle mutants and reconstructed ancestral state (double mutant). I have performed competition
experiments in LB Broth nutrient-enriched medium and M9 minimal (glucose as a single carbon
source). In EF4 and YebC proteins, I observed a valley of lower tness in single mutants in the
local tness landscape and restoration peak in the ancestral state. I report the rst experimen-
tal assessment of the prediction of coevolution within a protein. I have also observed a peak
in one of the single mutants of IspH candidates, which compensates for the charge only in one
direction of the mutation. We used direct measurement of tness estimation in E. coli rather
than using a proxy for tness, i.e., protein tness mentioned in most studies. The results of
this study provide experimental evidence of the compensating nature in two out of three tested
candidates, highlighting the potential of coevolution detection methods as tools to understand
molecular evolution. This study helps to understand the interaction of the amino acids in the
3D space and can be used to predict the mechanism of evolution.
