Dimerization of protein G B1 domain at low pH: a conformational switch caused by loss of a single hydrogen bond.

A number of signals in the NMR spectrum of the B1 domain of staphylococcal protein G (GB1) show a chemical shift dependence on the concentration of the protein at pH 3 but not at neutral pH, implying the existence of self-association at low pH. NMR backbone relaxation experiments show that GB1 undergoes a slow conformational exchange at pH 3, which is not seen at higher pH. Analysis of relaxation dispersion experiments yields a self-association constant of 50 mM, and shows that (15)N chemical shift changes in the dimer interface are up to 3 ppm. The shift changes measured from concentration-dependent HSQC spectra and from relaxation dispersion show good consistency. Measurements of chemical shifts as a function of pH show that a hydrogen bond between the sidechains of Asp44 and Gln40 is broken when Asp44 is protonated, and that loss of this hydrogen bond leads to the breaking of the (i, i + 4) backbone helical hydrogen bond from Asp44 HN to Gln40 O, and therefore to a loss of two residues from the C-terminal end of the helix. This weakens the helix structure and facilitates the loss of further helical structure thus permitting dimerization, which is suggested to occur in the same way as observed for the A42F mutant of GB1 (Jee et al., Proteins 2007;71:1420-1431), by formation of an antiparallel beta-sheet between the edge strands 2 in two monomers. The monomer/dimer ratio is thus a finely balanced equilibrium even in the wild type protein.