Bachhawat K, Kapoor M, Dam T K, Surolia A
Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
Biochemistry. 2001 Jun 19;40(24):7291-300. doi: 10.1021/bi0027783.
Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 if 2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (DeltaC(p) and also the temperature dependence of the thermodynamic parameters, namely, DeltaG, DeltaH, and DeltaS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters DeltaH(g), DeltaS(g), and DeltaC(p) as 174.1 kcal x mol(-1), 0.512 kcal x mol(-1) x K(-1), and 3.41 kcal x mol(-1) x K(-1), respectively, at T(g) = 339.4 K. Also, the free energy of unfolding, DeltaG(s), at its temperature of maximum stability (T(s) = 293 K) is 13.13 kcal x mol(-1). Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined DeltaC(p) (3.2 +/- 0.28 kcal x mol(-1) x K(-1)) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small DeltaC(p) for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.
大蒜凝集素（ASAI）是一种异源二聚体甘露糖特异性鳞茎凝集素，具有两条分子量分别为11.5 kDa和12.5 kDa的多肽链。通过差示扫描量热法和圆二色性对ASAI的热解折叠进行表征，结果表明其具有高度可逆性，可定义为一个两态过程，即折叠的二聚体直接转化为未折叠的单体（若2U则为A2）。利用热诱导和等温盐酸胍（GdnCl）诱导的解折叠，并分别通过差示扫描量热法（DSC）、远紫外圆二色性（far-UV CD）和荧光进行监测，确定了其构象稳定性与温度、GdnCl浓度和pH的关系。对这些数据的分析得出了解折叠时的热容变化（ΔC(p)）以及热力学参数ΔG、ΔH和ΔS的温度依赖性。将稳定性曲线拟合到修正的吉布斯 - 亥姆霍兹方程，分别得到了热力学参数ΔH(g)、ΔS(g)和ΔC(p)的估计值，在T(g) = 339.4 K时分别为174.1 kcal·mol⁻¹、0.512 kcal·mol⁻¹·K⁻¹和3.41 kcal·mol⁻¹·K⁻¹。此外，在其最大稳定性温度（T(s) = 293 K）下的解折叠自由能ΔG(s)为13.13 kcal·mol⁻¹。与迄今为止研究的大多数寡聚蛋白不同，该凝集素实验测定的ΔC(p)（3.2 ± 0.28 kcal·mol⁻¹·K⁻¹）与通过计算其可及表面积评估得到的结果之间表现出极好的一致性。这进而表明，无论变性方法如何，该蛋白都能达到完全未折叠的状态。不存在任何折叠中间体表明四级相互作用是该蛋白构象稳定性的主要贡献因素，这与其X射线结构密切相关。ASAI解折叠时较小的ΔC(p)反映出折叠的二聚体蛋白中埋藏的疏水核心相对较小。
