Nonetheless, NMR spectroscopy does provide some experimental avenues for characterizing membrane transporter structure and function. The hydrophobic nature of these proteins leads to aggregation that represents a barrier to crystallization similarly, the aggregation properties of these proteins in solution thwart efforts to collect high-quality NMR spectra. Unfortunately, ascertaining complete structures of these transporters currently poses a difficult challenge for the structural biologist, as whole membrane proteins are generally refractory to analyses by X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Knowledge of membrane transporter molecular structures is crucial for obtaining a detailed understanding of the mechanism by which these proteins shuttle their cargo across a biological membrane. In this chapter we discuss some of these methods, including dynamic filtering, comparison of 1H- 15N HSQC peaks’ intensities, transverse relaxation time, measurements of 1H- 15N nuclear Overhauser effect (NOE) values, and measurements of T 1ρ relaxation time. Several NMR methods were developed based on these characteristics. Their mobility will resemble that of the antibody’s residues. The interacting peptide residues become considerably immobilized upon binding. Epitope mapping by NMR is based on the difference in mobility between the amino acid residues of a peptide antigen that interact tightly with the antibody and residues outside the epitope that do not interact with the antibody. Choice of the specific method depends upon the dissociation constant, especially the ligand off-rate. NMR can be used to study antibodies in complexes that exhibit a wide range of binding affinities from very weak and transient to very tight. Nuclear magnetic resonance (NMR) is a very powerful tool for determining the boundaries of peptide epitopes recognized by antibodies. Here, we describe general implementation and latest improvements of expressed protein ligation method for the production of segmental isotopic labeled NMR samples. By utilizing intein techniques (Wood and Camarero, J Biol Chem 289:14512–14519, 2014 Paulus, Annu Rev Biochem 69:447–496, 2000), two related approaches can generally be used in the segmental isotopic labeling of proteins: expressed protein ligation (Muir, Annu Rev Biochem 72:249–289, 2003) and protein trans-splicing (Shah et al., J Am Chem Soc 134:11338–11341, 2012). Advantages of segmental isotopic labeling include selective examination of specific segment(s) within a protein by NMR, significantly reducing the spectral complexity for large proteins, and allowing for the application of a variety of solution-based NMR strategies. The procedure used here is applicable for the characterization of cell-wall components in various plant biomasses.Segmental isotopic labeling of samples for NMR studies is attractive for large complex biomacromolecular systems, especially for studies of function-related protein–ligand interactions and protein dynamics (Goto and Kay, Curr Opin Struct Biol 10:585–592, 2000 Rosa et al., Molecules (Basel, Switzerland) 18:440, 2013 Hiroaki, Expert Opin Drug Discovery 8:523–536, 2013). Characterization of the NMR signals for the whole cell-wall components, including lignin, cellulose, and hemicelluloses, was achieved by comparison with isolated lignin and commercial cellulose and hemicelluloses (arabinoxylan, galactomannan, and glucomannan). The acetylated lignin and polysaccharide signals dispersed reasonably well on the 2D spectra. (1)H-(13)C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments were successfully conducted with the acetylated woods in dimethyl sulfoxide (DMSO)-d(6). The dissolved woods were then subjected to in situ acetylation, and the fully acetylated woods were regenerated from Cl. Milled woods prepared by planetary ball-milling for 8 h dissolved completely in Cl at 100 ☌ in 2 h. An ionic liquid, 1-butyl-3-methylimidazolium chloride (Cl), was used to dissolve Japanese fir (Abies sachallnensis MAST) wood.
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