Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA.
Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA; Division of Hematology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA.
J Mol Biol. 2014 Jan 23;426(2):347-61. doi: 10.1016/j.jmb.2013.10.016. Epub 2013 Oct 22.
Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis-trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans-cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH2; reactions become slower and more stochastic as the pH increases up to pH7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.
轻链淀粉样变性是一种破坏性疾病，其中免疫球蛋白轻链形成淀粉样纤维，导致器官功能障碍和死亡。以前的研究表明，在某些与轻链淀粉样变性相关的蛋白质中，蛋白质热力学稳定性与淀粉样形成倾向之间存在直接相关性。在这里，我们研究了体细胞突变对蛋白质稳定性的影响，以及与野生型种系控制蛋白相比，AL-103 单复突变体和双复突变体在体外形成纤维的影响。我们观察到所有蛋白质的热变性和复性都存在扫描速率依赖性和滞后现象。这表明，尽管该过程在实验上是可逆的，但折叠/展开反应是由动力学决定的，具有不同的展开和折叠动力学常数。我们对 AL-103 和 AL-103 delP95aIns 的结构分析表明，折叠/展开过程与顺式-反式脯氨酸异构化存在动力学偶联。我们的数据表明，缺失脯氨酸 95a（AL-103 delP95aIns），消除了患者蛋白 AL-103 中存在的反式-顺式二脯氨酸基序，导致热力学稳定性显著增加，并且与 AL-103 相比，纤维形成动力学显著延迟。纤维形成是 pH 值依赖性的；所有蛋白质在 pH2 下形成纤维；随着 pH 值增加到 pH7，反应变得更慢且更随机。基于这些结果，我们提出，除了热力学稳定性外，动力学稳定性（可能受顺式脯氨酸 95a 的存在影响）在 AL-103 淀粉样纤维形成过程中起主要作用。
