Laboratory of Biochemstry and Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto, Kagoshima, 890-0065, Japan.
Protein Sci. 2013 Nov;22(11):1582-91. doi: 10.1002/pro.2359. Epub 2013 Sep 30.
Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10-20% 2,2,2-trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.
嗜盐蛋白的特点是净负电荷高，疏水性氨基酸比例相对较小，因此不易聚集。本研究中使用的中盐嗜盐菌 Chromohalobacter salexigens 中的组氨酸丰富的金属结合蛋白 (HP) 也具有这些特性。在这里，我们研究了嗜盐蛋白如何在体外形成淀粉样纤维。His 标记的 HP 在 pH 2.0 和 58°C 下孵育时，很容易形成淀粉样纤维，如硫黄素荧光、CD 光谱和透射或原子力显微镜观察到的那样。然而，在这些低 pH 苛刻条件下，His-HP 很快被水解成更小的肽，这些肽很可能是导致淀粉样纤维快速形成的原因。从纤维中分离出的三个主要酸水解肽很容易形成纤维。Waltz 软件预测这些肽序列中的肽易于形成纤维，合成的肽也形成纤维。当在 pH 7.8 和 25°C 下用 10-20%的 2,2,2-三氟乙醇孵育全长 His-HP 时，也无需肽键断裂即可轻易形成淀粉样纤维。
