Lin T Y, Kim P S
Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center 02142.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10573-7. doi: 10.1073/pnas.88.23.10573.
For proteins that contain a disulfide bond, stability is linked thermodynamically to thiol-disulfide exchange. We use this relationship to obtain unfolding free energies for both the reduced and oxidized forms of Escherichia coli thioredoxin from measurements of the effective concentrations of protein thiols. We then evaluate the effect of an amino acid substitution on disulfide bond formation in both the native and denatured states of the protein. Although the Pro-34----Ser substitution in thioredoxin results in a decrease of the effective concentration of protein thiols in the native state, the effective concentration increases in the denatured state. The net effect of the amino acid substitution is to increase the stability of reduced thioredoxin by approximately 2.4 kcal/mol, whereas the stability of the oxidized protein remains the same. By assuming a two-state unfolding equilibrium and a mutation free energy of -7.7 kcal/mol for the Pro-34----Ser substitution in the reduced, urea-unfolded state (based on estimates of solvation and entropic changes), we obtained relative free energies for the native and denatured states of the mutant and wild-type proteins, in both the reduced and oxidized forms.
对于含有二硫键的蛋白质，其稳定性在热力学上与硫醇-二硫键交换相关。我们利用这种关系，通过测量蛋白质硫醇的有效浓度，获得了大肠杆菌硫氧还蛋白还原态和氧化态的解折叠自由能。然后，我们评估了氨基酸取代对蛋白质天然态和变性态中二硫键形成的影响。尽管硫氧还蛋白中Pro-34突变为Ser导致天然态下蛋白质硫醇的有效浓度降低，但在变性态下有效浓度增加。氨基酸取代的净效应是使还原态硫氧还蛋白的稳定性增加约2.4千卡/摩尔，而氧化态蛋白质的稳定性保持不变。通过假设在还原的、尿素变性状态下Pro-34突变为Ser的双态解折叠平衡和突变自由能为-7.7千卡/摩尔（基于溶剂化和熵变的估计），我们获得了突变型和野生型蛋白质在还原态和氧化态下天然态和变性态的相对自由能。
