title: Rational stabilization of complex proteins: a divide and combine approach.
abstract: Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones).
authors.affiliation: 1] Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain [2] Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Lamazares
authors.affiliation: 1] Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain [2] Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Clemente
authors.affiliation: 1] Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain [2] Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Bueno
authors.affiliation: 1] Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain [2] Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain [3] Fundación ARAID, Gobierno de Aragón, Spain.
authors.forename: Adrián
authors.initials: A
authors.lastname: Velázquez-Campoy
authors.affiliation: 1] Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza, Zaragoza, Spain [2] Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Sancho
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005418:Flavodoxin / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D005418:Flavodoxin; D046912:Multiprotein Complexes; D011506:Proteins; D011994:Recombinant Proteins; C026932:apoflavodoxin