An in-vitro selection strategy was used to obtain strongly stabilized variants of the β1 domain of protein G (Gβ1). In a two-step approach, first candidate positions with a high potential for stabilization were identified in Gβ1 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 Gβ1 molecule. This variant with the four mutations E15V, T16L, T18I, and N37L showed an increase of 35.1 °C in the transition midpoint and of 28.5 kJ mol−1 (at 70 °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.
Submitter: Marie Ary
Submission Date: Feb. 20, 2017, 4:26 p.m.
|Number of data points||114|
|Assays/Quantities/Protocols||Experimental Assay: ΔGD (30°C) ; Experimental Assay: ΔGD (70°C) ; Experimental Assay: [GdmCl]m ; Experimental Assay: ΔHD(Tm) ; Experimental Assay: Tm pH7.0 ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0 ; Experimental Assay: m ; Derived Quantity: ΔΔGD (70°C) ; Derived Quantity: ΔΔGD (30°C)|
|Libraries||GdmCl unfolding of best site directed mutagenesis variants (Table 4) ; Thermal stabilities of variants selected by saturation mutagenesis of 5 positions (Table 3) ; Proside selected variants generated by EP-PCR (Table 1) ; Comparison of variants from in vitro evolution or from CPD (Table 5)|