title: Additive effect of single amino acid replacements on the kinetic stability of β-glucosidase B.
abstract: Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of β-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG ( N→TS )) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C ( β ) s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.
authors.affiliation: Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, 09340, Mexico, D. F., Mexico.
authors.forename: Menandro
authors.initials: M
authors.lastname: Camarillo-Cadena
authors.forename: Gerogina
authors.initials: G
authors.lastname: Garza-Ramos
authors.forename: Mariana
authors.initials: M
authors.lastname: Peimbert
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
authors.forename: Gerardo
authors.initials: G
authors.lastname: Pérez-Hernández
authors.forename: Rafael A
authors.initials: RA
authors.lastname: Zubillaga
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase