Division of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
Biochemistry. 2012 Feb 14;51(6):1269-80. doi: 10.1021/bi2013799. Epub 2012 Feb 3.
Protein self-association and protein unfolding are two temperature-dependent processes whose understanding is of utmost importance for the development of biological pharmaceuticals because protein association may stabilize or destabilize protein structure and function. Here we present new theoretical and experimental methods for analyzing the thermodynamics of self-association and unfolding. We used isothermal dilution calorimetry and analytical ultracentrifugation to measure protein self-association and introduced binding partition functions to analyze the cooperative association equilibria. In a second type of experiment, we monitored thermal protein unfolding with differential scanning calorimetry and circular dichroism spectroscopy and used the Zimm−Bragg theory to analyze the unfolding process. For α-helical proteins, the cooperative Zimm−Bragg theory appears to be a powerful alternative to the classical two-state model. As a model protein, we chose highly purified human recombinant apolipoprotein A-I. Self-association of Apo A-I showed a maximum at 21 °C with an association constant Ka of 5.6 × 10(5) M(−1), a cooperativity parameter σ of 0.003, and a maximal association number n of 8. The association enthalpy was linearly dependent on temperature and changed from endothermic at low temperatures to exothermic above 21 °C with a molar heat capacity ΔC(p)° of −2.76 kJ mol(−1) K(−1). Above 45 °C, the association could no longer be measured because of the onset of unfolding. Unfolding occurred between 45 and 65 °C and was reversible and independent of protein concentration up to 160 μM. The midpoint of unfolding (T(0)) as measured by DSC was 52−53 °C; the enthalpy of unfolding (ΔH(N)(U)) was 420 kJ/mol. The molar heat capacity (Δ(N)(U)C(p)) increased by 5.0 ± 0.5 kJ mol(−1) K(−1) upon unfolding corresponding to a loss of 80−85 helical segments, which was confirmed by circular dichroism spectroscopy. Unfolding was highly cooperative with a nucleation parameter σ of 4.4 × 10(−5).
蛋白质自组装和蛋白质变性是两个依赖温度的过程，对于生物制药的发展至关重要，因为蛋白质的聚集可能会稳定或破坏蛋白质的结构和功能。在这里，我们提出了新的理论和实验方法来分析自组装和变性的热力学。我们使用等温稀释量热法和分析超速离心法来测量蛋白质的自组装，并引入结合分配函数来分析协同结合平衡。在第二类实验中，我们通过差示扫描量热法和圆二色光谱法监测热蛋白变性，并使用 Zimm-Bragg 理论来分析变性过程。对于α-螺旋蛋白，协同 Zimm-Bragg 理论似乎是经典二态模型的有力替代。作为模型蛋白，我们选择了高度纯化的人重组载脂蛋白 A-I。Apo A-I 的自组装在 21°C 时达到最大值，结合常数 Ka 为 5.6×10(5)M(−1)，协同参数σ为 0.003，最大结合数 n 为 8。结合焓与温度呈线性关系，在低温下为吸热，在 21°C 以上为放热，摩尔热容ΔC(p)°为−2.76 kJ mol(−1) K(−1)。在 45°C 以上，由于变性的开始，无法再测量到结合。在 45-65°C 之间发生变性，并且在高达 160μM 的蛋白质浓度下是可逆且独立的。由 DSC 测量的变性中点（T(0)）为 52-53°C；变性焓（ΔH(N)(U)）为 420kJ/mol。摩尔热容（Δ(N)(U)C(p)）在变性时增加了 5.0±0.5 kJ mol(−1) K(−1)，对应于 80-85 个螺旋段的丢失，这通过圆二色光谱法得到了证实。变性具有高度协同性，成核参数σ为 4.4×10(−5)。
