Journal
BIOCHEMISTRY
Volume 54, Issue 28, Pages 4307-4319Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.5b00506
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Funding
- NIH [R01 GM074134, GM068928, RR11969, RR16649]
- NRSA T32 institutional training grants [GM008759, GM065103]
- National Science Foundation [9602941, 0230966]
- W. M. Keck Foundation
- Div Of Biological Infrastructure
- Direct For Biological Sciences [9602941, 0230966] Funding Source: National Science Foundation
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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 C-13/N-15-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 C-13 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 (CH3)-C-13-H-1-labeled methyls by comparing distance information obtained from three-dimensional methyl methyl 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 methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed NOE-methyl cluster 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 (CH3)-C-13-H-1-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.
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