信号肽与DmsD结合的物理性质。

Physical nature of signal peptide binding to DmsD.

作者信息

Winstone Tara L, Workentine Matthew L, Sarfo Kwabena J, Binding Andrew J, Haslam Bronwyn D, Turner Raymond J

机构信息

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4.

出版信息

Arch Biochem Biophys. 2006 Nov 1;455(1):89-97. doi: 10.1016/j.abb.2006.08.009. Epub 2006 Aug 22.

DOI:10.1016/j.abb.2006.08.009

Abstract

Here we describe the biophysical characterization of the interaction of the redox enzyme maturation protein DmsD with the signal peptide of its target protein, DmsA. Isothermal titration calorimetry (ITC), size exclusion chromatography (SEC), and an in vitro Far-Western assay is used to show that DmsD binds the twin-arginine signal peptide from DmsA in the micromolar range and in a 1:1 molar ratio. The SEC also shows that there is no oligomerization upon binding. Urea and guanidium hydrochloride denaturation profiles demonstrate the stability of DmsD and give insights on how electrostatic and hydrophobic interactions are important within this binding process. Furthermore, by use of N- and C-terminal fusions of DmsA signal peptide to GST, we observe that N-terminal display of the peptide is important for binding DmsD. In addition, all the folding forms of DmsD were found to bind the DmsA signal peptide as observed with the Far-Western assay.

摘要

在此，我们描述了氧化还原酶成熟蛋白DmsD与其靶蛋白DmsA的信号肽之间相互作用的生物物理特性。等温滴定量热法（ITC）、尺寸排阻色谱法（SEC）和体外Far-Western分析表明，DmsD以微摩尔浓度范围和1:1的摩尔比结合来自DmsA的双精氨酸信号肽。SEC还表明，结合后不会发生寡聚化。尿素和盐酸胍变性曲线证明了DmsD的稳定性，并深入了解了静电和疏水相互作用在该结合过程中的重要性。此外，通过使用DmsA信号肽与GST的N端和C端融合体，我们观察到该肽的N端展示对于结合DmsD很重要。此外，如Far-Western分析所观察到的，发现DmsD的所有折叠形式均能结合DmsA信号肽。