Wilcock D, Pisabarro M T, López-Hernandez E, Serrano L, Coll M
Departament de Biologia Molecular i Cel.lular, Centre d'Investigació i Desenvolupament-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain.
Acta Crystallogr D Biol Crystallogr. 1998 May 1;54(Pt 3):378-85. doi: 10.1107/s0907444997012158.
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.
已分别测定了信号转导蛋白CheY的两个双突变体（F14N/V21T和F14N/V86T）的晶体结构，分辨率分别为2.4 Å和2.2 Å。结构通过分子置换法解析，最终精修的R值分别为18.4%和19.2%。结合尿素变性实验，这些结构已被用于分析疏水残基被能够形成氢键的残基取代时突变的影响。突变Phe14Asn稳定性的大幅增加（-12.1 kJ mol-1）源于两个因素：反向疏水效应和在α-螺旋1处形成良好的N-帽。此外，还发现了一种前后氢键模式，类似于N-帽盒，涉及Asn14和Arg18。疏水核心处的两个Val到Thr突变具有不同的热力学效应：突变Val21Thr不影响蛋白质的稳定性，而突变Val86Thr导致1.7 kJ mol-1的小程度去稳定化。在第21位，在α-螺旋1内部形成了一个向后的侧链到主链氢键，与i - 4残基的羰基O原子相连，而突变侧链没有移动。在核心引入极性基团的去稳定化效应通过形成额外的氢键得到有效补偿。在第86位，新的Oγ原子通过χ1旋转从疏水环境逸出到相邻的亲水腔中形成新的氢键。在这种情况下，等排的Val到Thr取代具有破坏作用，但稳定能的损失部分通过形成氢键得到补偿。本文描述的两个晶体结构强调了氢键成分对蛋白质稳定性的重要性。
