Dimer Formation of a Stabilized Gβ1 Variant: A Structural and Energetic Analysis

In previous work, a strongly stabilized variant of the beta 1 domain of streptococcal protein G (G beta 1) was obtained by an in vitro selection method. This variant, termed G beta 1-M2, contains the four substitutions E15V, T16L, T18I, and N37L. Here we elucidated the molecular basis of the observed strong stabilizations. The contributions of these four residues were analyzed individually and in various combinations, additional selections with focused G beta 1 gene libraries were performed, and the crystal structure of G beta 1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type G beta 1 by contributions of between 1.6 and 6.0 kJ mol(-1) (at 70 degrees C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of G beta 1. They also increase the tendency to form dimers, as shown by dependence on the concentration of apparent molecular mass in analytical ultracentrifugation, by concentration-dependent stability, and by a strongly increased van't Hoff enthalpy of unfolding. The 0.88-angstrom crystal structure of G beta 1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two G beta 1-M2 molecules via six intermolecular hydrogen bonds between the two p strands 2 and 2' and an adjacent self-complementary hydrophobic surface area, which is created by the T16L and N37L substitutions and a large 120 degrees rotation of the Tyr33 side chain. This removal of hydrophilic groups and the malleability of the created hydrophobic surface provide the basis for the dimer formation of stabilized G beta 1 variants. (C) 2009 Elsevier Ltd. All rights reserved.