Biochemical characterization of paralyzed flagellum proteins A (PflA) and B (PflB) from Helicobacter pylori flagellar motor.

IF 3.8 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Bioscience Reports Pub Date : 2024-09-25 DOI:10.1042/BSR20240692
Xiaotian Zhou, Muhammad F Khan, Yue Xin, Kar L Chan, Anna Roujeinikova
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Abstract

Motility by means of flagella plays an important role in the persistent colonization of Helicobacter pylori in the human stomach. The H. pylori flagellar motor has a complex structure that includes a periplasmic scaffold, the components of which are still being identified. Here, we report the isolation and characterization of the soluble forms of two putative essential H. pylori motor scaffold components, proteins PflA and PflB. We developed an on-column refolding procedure, overcoming the challenge of inclusion body formation in Escherichia coli. We employed mild detergent sarkosyl to enhance protein recovery and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO)-containing buffers to achieve optimal solubility and monodispersity. In addition, we showed that PflA lacking the β-rich N-terminal domain is expressed in a soluble form, and behaves as a monodisperse monomer in solution. The methods for producing the soluble, folded forms of H. pylori PflA and PflB established in this work will facilitate future biophysical and structural studies aimed at deciphering their location and their function within the flagellar motor.

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幽门螺旋杆菌鞭毛运动中的麻痹鞭毛蛋白 A(PflA)和 B(PflB)的生化特征。
幽门螺旋杆菌通过鞭毛运动,在人类胃部持续定殖中发挥着重要作用。幽门螺杆菌鞭毛运动具有复杂的结构,包括一个外质支架,其成分仍在鉴定中。在此,我们报告了两种推定的幽门螺杆菌鞭毛马达支架基本成分--蛋白质 PflA 和 PflB 的可溶形式的分离和表征。我们开发了一种柱上重折叠程序,克服了在大肠杆菌中形成包涵体的难题。我们采用了温和的去垢剂 Sarkosyl 来提高蛋白质的回收率,并使用含有正十二烷基-N,N-二甲胺-N-氧化物(LDAO)的缓冲液来达到最佳的溶解度和单分散性。此外,我们还发现,缺乏富含 β N 端结构域的 PflA 可以以可溶形式表达,并在溶液中表现为单分散单体。这项工作中建立的生产幽门螺杆菌 PflA 和 PflB 的可溶性折叠形式的方法将有助于未来的生物物理和结构研究,以破译它们在鞭毛马达中的位置和功能。
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来源期刊
Bioscience Reports
Bioscience Reports 生物-细胞生物学
CiteScore
8.50
自引率
0.00%
发文量
380
审稿时长
6-12 weeks
期刊介绍: Bioscience Reports provides a home for sound scientific research in all areas of cell biology and molecular life sciences. Since 2012, Bioscience Reports has been fully Open Access and publishes all papers under the liberal CC BY licence, giving the life science community quality research to share and discuss.Content before 2012 is subscription-only, and is accessible via archive purchase. Articles are assessed on soundness, providing a home for valid findings and data. We welcome papers that span disciplines (e.g. chemistry, medicine), including papers describing: -new methodologies -tools and reagents to probe biological questions -mechanistic details -disease mechanisms -metabolic processes and their regulation -structure and function -bioenergetics
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