Carra J H, Murphy E C, Privalov P L
Department of Biology and Biocalorimetry Center, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biophys J. 1996 Oct;71(4):1994-2001. doi: 10.1016/S0006-3495(96)79397-9.
We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.
我们使用差示扫描量热法和圆二色性测量研究了T4溶菌酶显著去稳定化突变体形式的折叠情况。此前观察到，与一种假野生型参考蛋白相比，该蛋白N端区域一个α螺旋中的三个突变，即丙氨酸插入突变S44[A]和K48[A]以及替换突变A42K，导致了出乎意料的低表观熔解焓变。该假野生型参考蛋白以基本的两态方式发生热去折叠。然而，我们发现这三种突变蛋白的去折叠协同性降低，这部分解释了它们较低的表观焓变。需要一个包含离散中间体的三态去折叠模型来描述突变蛋白的熔解。为了准确计算折叠的能量变化，必须考虑协同性的降低。分两个阶段去折叠反映了溶菌酶蛋白家族潜在的两个亚结构域结构。
