Dragan Anatoly I, Potekhin Sergey A, Sivolob Andrei, Lu Min, Privalov Peter L
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 2004 Nov 30;43(47):14891-900. doi: 10.1021/bi048365+.
Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.
采用圆二色光谱、动态光散射和扫描量热法等动力学和热力学方法，研究了温度诱导的三链α-螺旋卷曲螺旋Lpp-56的可逆解折叠和重折叠过程。发现在100 mM NaCl存在下，该蛋白在中性溶液中的解折叠和重折叠反应均具有异常缓慢的动力学特征，这使得能够详细研究这些反应的机制。动力学分析表明，这种卷曲螺旋的解折叠是一个单阶段一级反应，而重折叠是一个单阶段三级反应。解折叠的活化焓和熵对温度的依赖性不明显，且均显著大于折叠反应的活化焓和熵，后者对温度有显著依赖性。解折叠反应的活化热容量变化接近零，而折叠反应的活化热容量变化则相当显著。从Lpp-56卷曲螺旋获得的活化参数与结构参数之间的相关性表明，在过渡态中螺旋间范德华相互作用被破坏，尽管过渡态仍然紧密，且水尚未渗透到界面中；从瞬态到解折叠态的转变导致界面处暴露的非极性基团水化以及螺旋的破坏。Lpp-56链折叠和缔合的倾向较低是由中性pH下大量带电基团引起的。一方面，这些电荷产生相当大的排斥力，使链的螺旋构象不稳定。另一方面，它们使折叠的螺旋平行且对齐，使得非极性面彼此相对，且带相反电荷的基团可能形成盐键，这对于三聚体卷曲螺旋的形成很重要。pH的降低消除了盐键，显著降低了Lpp-56的稳定性；其结构变得不那么刚性，解折叠速度也快得多。
