title: In vitro evolution of a hyperstable Gbeta1 variant.
abstract: An in-vitro selection strategy was used to obtain strongly stabilized variants of the beta1 domain of protein G (Gbeta1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gbeta1 libraries that were created by error-prone PCR, and then, after randomization of these positions by saturation mutagenesis, strongly stabilized variants were selected. For both steps the in-vitro selection method Proside was employed. Proside links the stability of a protein with the infectivity of a filamentous phage. Ultimately, residues from the two best selected variants were combined in a single Gbeta1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 degrees C in the transition midpoint and of 28.5 kJ mol(-1) (at 70 degrees C) in the Gibbs free energy of stabilization. It was considerably more stable than the best variant from a previous Proside selection, in which positions were randomized that had originally been identified by computational design. Only a single substitution (T18I) was found in both selections. The best variants from the present selection showed a higher cooperativity of thermal unfolding, as indicated by an increase in the enthalpy of unfolding by about 60 kJ mol(-1). This increase is apparently correlated with the presence of Leu residues that were selected at the positions 16 and 37.
authors.affiliation: Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wunderlich
authors.forename: Franz X
authors.initials: FX
authors.lastname: Schmid
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D019143:Evolution, Molecular; D006358:Hot Temperature; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011897:Random Allocation
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus