Tischer Alexander, Machha Venkata R, Rösgen Jörg, Auton Matthew
Division of Hematology, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota.
Department Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania, 17033.
Biopolymers. 2018 Aug;109(8):e23106. doi: 10.1002/bip.23106. Epub 2018 Feb 19.
Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how and the urea m-value interconvert through the slope of c versus T, . This relationship permits the calculation of at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.
蛋白质相图具有独特的潜力，即使从用于跟踪蛋白质解折叠转变的实验观测值中不容易看出非二态特征，也能识别出额外热力学状态的存在。血管性血友病因子A3结构域的二态分析先前已揭示，与通过线性外推法获得的自由能的吉布斯 - 亥姆霍兹分析相比，热解折叠转变获得的量热焓存在差异（Tischer和Auton，《蛋白质科学》2013年；22(9):1147 - 60）。我们使用尿素 - 温度相图的克劳修斯 - 克拉佩龙分析解决了这个热力学难题，该分析定义了如何通过c对T的斜率，即 ，使 和尿素m值相互转换。这种关系允许从通过等温尿素变性获得的m值计算低温下的 ，以及从通过等尿素热变性获得的 计算高温下的m值。随着温度升高，应用该方程揭示了两个协同性参数中的S形转变。 和m值的这种残余热协同性证实了存在一种额外状态，经证实这是由尿素膨胀态和热紧凑变性态之间的协同相变导致的。完整二硫键和羧酰胺化A3结构域的膨胀态和紧凑变性态集合之间的平衡比较表明，引入单个二硫键交联不会影响额外变性态的存在。然而，随着温度升高和尿素浓度降低，它确实对协同变性态坍塌转变做出了小的热力学有利自由能贡献（约 - 13 ± 1 kJ/mol）。变性态这种“协同坍塌”的热力学在对总自由能的焓和熵贡献之间保留了显著的补偿。
