title: Exploring the Structure-Function Loop Adaptability of a (β/α)(8)-Barrel Enzyme through Loop Swapping and Hinge Variability.
abstract: Evolution of proteins involves sequence changes that are frequently localized at loop regions, revealing their important role in natural evolution. However, the development of strategies to understand and imitate such events constitutes a challenge to design novel enzymes in the laboratory. In this study, we show how to adapt loop swapping as semiautonomous units of functional groups in an enzyme with the (β/α)(8)-barrel and how this functional adaptation can be measured in vivo. To mimic the natural mechanism providing loop variability in antibodies, we developed an overlap PCR strategy. This includes introduction of sequence diversity at two hinge residues, which connect the new loops with the rest of the protein scaffold, and we demonstrate that this is necessary for a successful exploration of functional sequence space. This design allowed us to explore the sequence requirements to functional adaptation of each loop replacement that may not be sampled otherwise. Libraries generated following this strategy were evaluated in terms of their folding competence and their functional proficiency, an observation that was formalized as a Structure-Function Loop Adaptability value. Molecular details about the function and structure of some variants were obtained by enzyme kinetics and circular dichroism. This strategy yields functional variants that retain the original activity at higher frequencies, suggesting a new strategy for protein engineering that incorporates a more divergent sequence exploration beyond that limited to point mutations. We discuss how this approach may provide insights into the mechanism of enzyme evolution and function.
authors.affiliation: Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Cuernavaca, C.P. 62210, México.
authors.forename: Adrián
authors.initials: A
authors.lastname: Ochoa-Leyva
authors.forename: Francisco
authors.initials: F
authors.lastname: Barona-Gómez
authors.forename: Gloria
authors.initials: G
authors.lastname: Saab-Rincón
authors.forename: Karina
authors.initials: K
authors.lastname: Verdel-Aranda
authors.forename: Filiberto
authors.initials: F
authors.lastname: Sánchez
authors.forename: Xavier
authors.initials: X
authors.lastname: Soberón
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic
chemical_list: D029968:Escherichia coli Proteins; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase