Structure analysis of two CheY mutants: importance of the hydrogen-bond contribution to protein stability.

The crystal structures of two double mutants (F14N/V21T and F14N/V86T) of the signal transduction protein CheY have been determined to a resolution of 2.4 and 2.2 A, respectively. The structures were solved by molecular replacement and refined to final R values of 18.4 and 19.2%, respectively. Together with urea-denaturation experiments the structures have been used to analyse the effects of mutations where hydrophobic residues are replaced by residues capable of establishing hydrogen bonds. The large increase in stabilization (-12.1 kJ mol-1) of the mutation Phe14Asn arises from two factors: a reverse hydrophobic effect and the formation of a good N-cap at alpha-helix 1. In addition, a forward-backward hydrogen-bonding pattern, resembling an N-capping box and involving Asn14 and Arg18, has been found. The two Val to Thr mutations at the hydrophobic core have different thermodynamic effects: the mutation Val21Thr does not affect the stability of the protein while the mutation Val86Thr causes a small destabilization of 1.7 kJ mol-1. At site 21 a backward side chain-to-backbone hydrogen bond is formed inside alpha-helix 1 with the carbonyl O atom of the i - 4 residue without movement of the mutated side chain. The destabilizing effect of introducing a polar group in the core is efficiently compensated for by the formation of an extra hydrogen bond. At site 86 the new Ogamma atom escapes from the hydrophobic environment by a chi1 rotation into an adjacent hydrophilic cavity to form a new hydrogen bond. In this case the isosteric Val to Thr substitution is disruptive but the loss in stabilization energy is partly compensated by the formation of a hydrogen bond. The two crystal structures described in this work underline the significance of the hydrogen-bond component to protein stability.