Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2

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• 作者：Yao Xiao ; Lisa R. Warner ; Michael P. Latham ; Natalie G. Ahn ; Arthur Pardi
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：July 21, 2015
• 年：2015
• 卷：54
• 期：28
• 页码：4307-4319
• 全文大小：694K
• ISSN：1520-4995

文摘

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly ¹³C/¹⁵N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for ¹³C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of ¹³C¹H₃-labeled methyls by comparing distance information obtained from three-dimensional methyl鈥搈ethyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl鈥搈ethyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed 鈥淣OE-methyl cluster鈥?onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV ¹³C¹H₃-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.