title: Relative tolerance of mesostable and thermostable protein homologs to extensive mutation.
abstract: Evolvability, designability, and plasticity of a protein are properties that are important to protein engineers, but difficult to quantify. Here, we directly compare homologous AroQ chorismate mutases from the thermophile Methanococcus jannaschii and the mesophile Escherichia coli with respect to their capacity to accommodate extensive mutation. The N-terminal helix comprising about 40% of these proteins was randomized at the genetic level using a binary pattern of hydrophobic and hydrophilic residues based on the respective wild-type sequences. Catalytically active library members were identified by a survival-selection assay in a chorismate mutase-deficient E. coli strain. Functional variants were found approximately approximately 10-times more frequently with the thermostable protein compared to its mesostable counterpart. Moreover, detailed sequence analysis revealed that functional M. jannaschii enzyme variants contained a smaller number of conserved residues and tolerated greater variability at individual sequence positions. Our results thus highlight the greater robustness of the thermostable protein with respect to amino acid substitution, while identifying specific sites important for constructing active enzymes. Overall, they support the notion that redesign projects will benefit from using a thermostable starting structure, even at very high mutational loads.
authors.affiliation: Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland.
authors.forename: Werner
authors.initials: W
authors.lastname: Besenmatter
authors.forename: Peter
authors.initials: P
authors.lastname: Kast
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002826:Chorismate Mutase / Q000737:chemistry / Q000235:genetics*; D015195:Drug Design; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D015723:Gene Library; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017354:Point Mutation; D011489:Protein Denaturation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013045:Species Specificity; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D002826:Chorismate Mutase