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The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-11-04 , DOI: 10.1016/j.bbagen.2020.129780
D. Santorelli , S. Rocchio , F. Fata , I. Silvestri , F. Angelucci , F. Imperi , D. Marasco , C. Diaferia , L. Gigli , N. Demitri , L. Federici , A. Di Matteo , C. Travaglini-Allocatelli

Background

Ribosome-binding factor A from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA) is a small ribosome assembly factor, composed by a single KH domain, involved in the maturation of the 30S subunit. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in a variety of cellular functions. However, although the ability of proteins to fold properly, to misfold or to aggregate is of paramount importance for their cellular functions, limited information is available on these dynamic properties in the case of KH domains.

Methods

PaRbfA thermodynamic stability and folding mechanism: Far-UV CD and fluorescence spectroscopy, stopped-flow kinetics and chevron plot analysis, site-directed mutagenesis. Fibrils characterization: FT-IR spectroscopy, Thioflavin T fluorescence, Transmission Electron Microscopy (TEM) and X-ray fibrils diffraction.

Results

Quantitative analysis of the (un)folding kinetics of PaRbfA show that, in vitro, the protein folds via a 3-states mechanism involving a transiently populated folding intermediate. We also provide experimental evidences that PaRbfA can form ordered fibrils endowed with cross-β structure even in mild conditions.

Conclusion

These results lead to the hypothesis that the folding intermediate of PaRbfA may expose (some of) the predicted amyloidogenic regions, which could act as aggregation nuclei in the fibrillogenesis.

General significance

The methodological approach presented herein could be readily adapted to verify the ability of other KH domain proteins to form cross-β structured fibrils and to transiently populate a folding intermediate.



中文翻译:

单个KH域蛋白的折叠和聚集特性:铜绿假单胞菌的核糖体结合因子A(RbfA)

背景

致病性细菌铜绿假单胞菌Pa RbfA)的核糖体结合因子A是一个小的核糖体装配因子,由单个KH结构域组成,参与30S亚基的成熟。这些结构域的特征是具有结合RNA或ssDNA的能力,通常位于参与多种细胞功能的蛋白质中。然而,尽管蛋白质正确折叠,错误折叠或聚集的能力对其细胞功能至关重要,但在KH域中,关于这些动态特性的信息有限。

方法

Pa RbfA热力学稳定性和折叠机理:远紫外CD和荧光光谱,停止流动力学和V形图分析,定点诱变。原纤维表征:FT-IR光谱,硫黄素T荧光,透射电子显微镜(TEM)和X射线原纤维衍射。

结果

Pa RbfA (非折叠)动力学的定量分析表明,在体外,该蛋白质通过涉及暂态折叠中间体的三态机制折叠。我们还提供实验证据,证明即使在温和条件下,Pa RbfA仍可形成具有交叉β结构的有序原纤维。

结论

这些结果导致了一个假说,即Pa RbfA的折叠中间体可能暴露(某些)预测的淀粉样蛋白生成区域,该区域可能在原纤维形成中充当聚集核。

一般意义

本文提出的方法学方法可以很容易地适用于验证其他KH域蛋白形成交叉β结构的原纤维并瞬时填充折叠中间体的能力。

更新日期:2020-11-19
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