Fuentes E J, Wand A J
Department of Chemistry, State University of New York at Buffalo 14260-3000, USA.
Biochemistry. 1998 Mar 17;37(11):3687-98. doi: 10.1021/bi972579s.
Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this "structured" molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry and hydrogen exchange. These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N- and C-terminal helices fold and unfold independently of the core of the molecule.
细胞色素b562是一种血红素结合蛋白，由四个螺旋折叠成经典的螺旋束基序。脱辅基细胞色素b562虽然保留了全蛋白的大部分拓扑结构，但却展现出通常与所谓蛋白质熔球相关的物理特征。在此，通过考察其氢交换行为对化学变性剂存在的依赖性，来探究这种“结构化”熔球的稳定性和动力学。与以这种方式研究的其他体系相比，脱辅基细胞色素b562展现出有限的氢交换速率动态范围，且分析需要开发一种定量方法。该蛋白质被发现具有三个亚全局协同稳定区域。最稳定的区域，即核心区域，由螺旋束的中间两个螺旋组成，而N端和C端螺旋具有独立且较低的稳定性。全局解折叠自由能对变性剂浓度的依赖性表明了一种结合模型的适用性，并解释了通过线性外推法获得的全局解折叠自由能与通过量热法和氢交换获得的全局解折叠自由能之间观察到的差异。这些观察结果对该蛋白质起作用的折叠途径类型施加了重大限制，并表明N端和C端螺旋独立于分子核心进行折叠和解折叠。
