In vitro evolution of a hyperstable Gbeta1 variant |
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Authors: | Wunderlich Michael Schmid Franz X |
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Institution: | Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universit?t Bayreuth, D-95440 Bayreuth, Germany. |
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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. |
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Keywords: | Gβ1 β1 domain of the streptococcal protein G G3P gene-3-protein of phage fd TM midpoint of a thermal unfolding transition GdmCl guanidinium chloride [GdmCl]m midpoint of a GdmCl-induced unfolding transition m cooperativity parameter of a GdmCl-induced unfolding transition m = ∂ΔGD/∂ [GdmCl] |
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