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


Abstract

In previous work, a strongly stabilized variant of the β1 domain of streptococcal protein G (Gβ1) was obtained by an in vitro selection method. This variant, termed Gβ1-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 Gβ1 gene libraries were performed, and the crystal structure of Gβ1-M2 was determined. All single substitutions (E15V, T16L, T18I, and N37L) stabilize wild-type Gβ1 by contributions of between 1.6 and 6.0 kJ mol− 1 (at 70 °C). Hydrophobic residues at positions 16 and 37 provide the major contribution to stabilization by enlarging the hydrophobic core of Gβ1. 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-Å crystal structure of Gβ1-M2 and NMR measurements in solution provide the explanation for the observed dimer formation. It involves a head-to-head arrangement of two Gβ1-M2 molecules via six intermolecular hydrogen bonds between the two β 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° 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 Gβ1 variants.

Submission Details

ID: CbMoCDHR

Submitter: Admin Admin

Submission Date: May 15, 2017, 5:06 p.m.

Version: 2

Publication Details
Thoms S;Max KE;Wunderlich M;Jacso T;Lilie H;Reif B;Heinemann U;Schmid FX,J Mol Biol (2009) Dimer formation of a stabilized Gβ1 variant: a structural and energetic analysis. PMID:19527728
Additional Information

Number of data points 305
Proteins Protein Gβ1
Unique complexes 19
Assays/Quantities/Protocols Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 4μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 10μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 108μM protein ; Experimental Assay: ΔHvH(monomer) 4μM protein ; Experimental Assay: ΔGD (70°C) ; Experimental Assay: [GdmCl]m of GB1-M2 at 0.25 μM protein ; Experimental Assay: m for GB1-M2 at 0.25μM protein ; Experimental Assay: ΔHvH(monomer) 1μM protein ; Experimental Assay: ΔHvH(monomer) 10μM protein ; Experimental Assay: ΔHvH(monomer) 108μM protein ; Experimental Assay: ΔHvH(monomer) 64μM protein ; Experimental Assay: ΔHvH(monomer) 62.2μM protein ; Experimental Assay: [GdmCl]m of GB1-M2 at 1.0 μM protein ; Experimental Assay: m for GB1-M2 at 10μM protein ; Experimental Assay: ΔHcal 108μM protein ; Experimental Assay: ΔHvH(monomer) 139μM protein ; Experimental Assay: ΔHvH(dimer) 1μM protein ; Experimental Assay: ΔHvH(dimer) 10μM protein ; Experimental Assay: ΔHcal 139μM protein ; Experimental Assay: ΔHcal 62.2μM protein ; Experimental Assay: m for GB1-M2 at 1.0μM protein ; Experimental Assay: ΔHvH(dimer) 4μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 1μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 64μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 62.2μM protein ; Experimental Assay: Tm in 1.5M GdmCl, pH7.0, 139μM protein ; Experimental Assay: ΔHcal 64μM protein ; Experimental Assay: [GdmCl]m of GB1-M2 at 10 μM protein ; Derived Quantity: ΔΔGD relative to WT ; Derived Quantity: ΔHvH/ΔHcal 64μM protein ; Derived Quantity: ΔHvH/ΔHcal 108μM protein ; Derived Quantity: ΔHvH/ΔHcal 62.2μM protein ; Derived Quantity: ΔHvH/ΔHcal 139μM protein
Libraries ΔHvH(dimer) for Gβ1-M2 at 10μM protein (Table 3) ; ΔHvH(dimer) for Gβ1-M2 at 4μM protein (Table 3) ; ΔHvH(dimer) for Gβ1-M2 at 1μM protein (Table 3) ; Cm and m for GB1-M2 at 1.0μM protein (Fig. S1) ; Cm and m for GB1-M2 at 10μM protein (Fig. S1) ; Tm, ΔHvH, ΔHcal, ΔHvH/ΔHcal for WT at 108 μM protein (Table 2) ; Tm, ΔHvH, ΔHcal, ΔHvH/ΔHcal for GB1-M2 at 139 μM protein (Table 2) ; Tm and enthalpy at 10 μM protein (Table 2, S1) ; Cm and m for GB1-M2 at 0.25μM protein (Fig. S1) ; Tm and enthalpy at 1 μM protein (Table 2, S1) ; Tm, ΔHvH, ΔHcal, ΔHvH/ΔHcal for GB1-M2 at 62.2 μM protein (Table 2) ; Tm, ΔHvH, ΔHcal, ΔHvH/ΔHcal for WT at 64 μM protein (Table 2) ; Individual contributions of mutations at positions 15,16,18, 33, 37 to stability of Gβ1-M2 (Table 1)
Sequence Assay Result Units