Jelesarov I, Bosshard H R
Biochemisches Institut, Universität Zürich, Switzerland.
J Mol Biol. 1996 Oct 25;263(2):344-58. doi: 10.1006/jmbi.1996.0579.
Folding thermodynamics of nine heterodimeric, parallel coiled coils were studied by isothermal titration calorimetry (ITC) and thermal unfolding circular dichroism measurements. The heterodimers were composed of an acidic and a basic 30-residue peptide, which when in isolation were monomeric and essentially unstructured. The reaction followed a two-state mechanism indicating that folding and association were coupled. delta Hfold, delta Sfold and delta Cp normalized per mol of residue were of the same magnitude as for monomeric globular proteins, hence the energetics of folding and association of the heterodimeric coiled coils was balanced similarly to the folding of a single polypeptide chain. Cavity creating Leu/Ala substitutions revealed strong and position-dependent energetic coupling between leucine residues in the hydrophobic core of the coiled coil. delta Gunfold (equivalent to -delta Gfold in the two-state reaction) was determined from thermal unfolding. Global stability curves were calculated according to the Gibbs-Helmholtz equation and using the combined free energy data from ITC and thermal unfolding. Maximum stabilities were between 15 and 37 degrees C and cold denaturation could be demonstrated by direct calorimetry. The stability curves were based on free energies of folding measured between 10 and 85 degrees C and under identical solvent conditions. This represents a novel experimental approach which circumvents the use of varying solvent conditions as is typically required to measure protein stability curves. Discrepancies were noticed between van't Hoff enthalpies deduced from thermal unfolding and measured by direct calorimetry. The discrepancies are thought to be due to residual ordered structure in the denatured single chains around room temperature but not near the transition midpoint temperature Tm. This demonstrates that over an extended temperature range the assumption of a common denatured state implicit in the van't Hoff analysis may not always be valid.
通过等温滴定量热法（ITC）和热变性圆二色性测量研究了九个异源二聚体平行卷曲螺旋的折叠热力学。这些异源二聚体由一个酸性和一个碱性的30个残基的肽组成，单独存在时它们是单体且基本无结构。该反应遵循两态机制，表明折叠和缔合是耦合的。每摩尔残基的ΔHfold、ΔSfold和ΔCp与单体球状蛋白的大小相同，因此异源二聚体卷曲螺旋的折叠和缔合能量学与单条多肽链的折叠相似，达到了平衡。造成空腔的亮氨酸/丙氨酸取代揭示了卷曲螺旋疏水核心中亮氨酸残基之间强烈且位置依赖的能量耦合。从热变性中确定了ΔGunfold（相当于两态反应中的-ΔGfold）。根据吉布斯-亥姆霍兹方程并使用ITC和热变性的组合自由能数据计算了全局稳定性曲线。最大稳定性在15至37摄氏度之间，并且可以通过直接量热法证明冷变性。稳定性曲线基于在10至85摄氏度之间以及相同溶剂条件下测量的折叠自由能。这代表了一种新颖的实验方法，避免了通常测量蛋白质稳定性曲线时所需的不同溶剂条件的使用。注意到从热变性推导并通过直接量热法测量的范特霍夫焓之间存在差异。这些差异被认为是由于室温附近变性单链中存在残留的有序结构，但在转变中点温度Tm附近则不存在。这表明在扩展的温度范围内，范特霍夫分析中隐含的共同变性状态的假设可能并不总是有效的。
