Function and solution structure of the Arabidopsis thaliana RALF8 peptide
Function and solution structure of the Arabidopsis thaliana RALF8 peptide
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DOI：
10.1002/pro.3628
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发表时间：
2019-06-01
期刊：
影响因子：
8
通讯作者：
Markley, John L.
中科院分区：
文献类型：
--
作者：
Frederick, Ronnie O.;Haruta, Miyoshi;Markley, John L.
我们报道了从大肠杆菌细胞中重组制备两个紧密相关的富含半胱氨酸的小分子植物多肽：快速碱化因子1(RALF1)和快速碱化因子8(RALF8)。RALF8天然序列的纯化样品显示出良好的核磁共振谱，并通过与植物受体激酶的相互作用、细胞质钙动员和体内根生长抑制而显示出生物活性。相比之下，RALF1只能作为包含N-末端组氨酸标签的结构物从包涵体中分离出来；它的核磁共振谱分辨率很低，表明存在聚集。我们制备了标记N-15和C-13的RALF8多肽样品用于核磁共振分析，获得了接近完整的H-1，C-13和N-15核磁共振归属，确定了其四个半胱氨酸残基的二硫键配对，并检查了其溶液结构。RALF8大部分是无序的，除了它的两个二硫键分别跨越的两个环路。
We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine-rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well-resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N-terminal His-tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with N-15 and C-13 for NMR analysis and obtained near complete H-1, C-13, and N-15 NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.