Schönbrunner N, Pappenberger G, Scharf M, Engels J, Kiefhaber T
Biozentrum der Universität Basel, Abteilung Biophysikalische Chemie, Klingelbergstrasse 70, CH 4056 Basel, Switzerland.
Biochemistry. 1997 Jul 22;36(29):9057-65. doi: 10.1021/bi970594r.
The role of preformed correct side chain interactions, such as disulfide bonds, on protein folding kinetics is still not well understood. We investigated the effect of disulfide bond replacements on folding and stability of the small beta-sheet protein tendamistat. Tendamistat folds very fast (tau = 10 ms at pH 7 in water) and without detectable intermediates, which facilitates molecular interpretation of the kinetic data. Tendamistat contains two disulfide bonds, one between cysteines 11 and 27, which connects the ends of a beta-hairpin, and a second one between cysteines 45 and 73, which brings together the two outer strands of a three-stranded beta-sheet. Two single-disulfide variants of the protein were prepared by site-directed mutagenesis (tendamistat C11A/C27S and tendamistat C45A/C73A), and the effects on stability and on folding were monitored. Replacement of either disulfide bond leads to a large decrease in protein stability (DeltaDeltaG0 = 6.0 kcal/mol for the C11A/C27S variant and 5.1 kcal/mol for the C45A/C73A variant). This effect is caused both by entropic stabilization of the unfolded state and by enthalpic destabilization of the native structure. Kinetic experiments show that the main effect of fixed side chain contacts is on the unfolding rate. For both single-disulfide variants, unfolding is strongly accelerated (4250 times in the C11A/C27S variant and 250 times in the C45A/C73A variant) whereas the refolding rate constants are only slightly decreased. The activation parameters show that the observed small effect on the refolding reaction in the C11A/C27S variant is a consequence of large and compensating changes in the entropy and enthalpy of activation. Structural interpretation of the kinetic data suggests that formation of the beta-hairpin stabilized by the C11-C27 disulfide bond forms in the rate-limiting step of the refolding process. The interactions between the outer strands of the beta-sheet connected by the C45-C73 disulfide bond, in contrast, are made late in refolding. These results support the idea that beta-hairpins are initiation sites for beta-sheet formation and that additional strands are added late in the folding process.
预先形成的正确侧链相互作用，如二硫键，在蛋白质折叠动力学中的作用仍未得到很好的理解。我们研究了二硫键替换对小β-折叠蛋白抑肽酶折叠和稳定性的影响。抑肽酶折叠非常快（在pH 7的水中，τ = 10毫秒）且没有可检测到的中间体，这便于对动力学数据进行分子解释。抑肽酶含有两个二硫键，一个在半胱氨酸11和27之间，连接β-发夹的两端，另一个在半胱氨酸45和73之间，将三链β-折叠的两条外侧链聚集在一起。通过定点诱变制备了该蛋白的两个单二硫键变体（抑肽酶C11A/C27S和抑肽酶C45A/C73A），并监测了其对稳定性和折叠的影响。任一二硫键的替换都会导致蛋白质稳定性大幅下降（C11A/C27S变体的ΔΔG0 = 6.0千卡/摩尔，C45A/C73A变体的ΔΔG0 = 5.1千卡/摩尔）。这种效应既是由未折叠状态的熵稳定化引起的，也是由天然结构的焓不稳定化引起的。动力学实验表明，固定侧链接触的主要影响在于解折叠速率。对于两个单二硫键变体，解折叠都被强烈加速（C11A/C27S变体中加速4250倍，C45A/C73A变体中加速250倍），而重折叠速率常数仅略有下降。活化参数表明，在C11A/C27S变体中观察到的对重折叠反应的微小影响是活化熵和活化焓的大的补偿性变化的结果。动力学数据的结构解释表明，由C11-C27二硫键稳定的β-发夹的形成发生在重折叠过程的限速步骤中。相比之下，由C45-C73二硫键连接的β-折叠外侧链之间的相互作用在重折叠后期形成。这些结果支持了β-发夹是β-折叠形成的起始位点且额外的链在折叠过程后期添加的观点。
