Amino Acid-Type Specific Labeling

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

If specific amino acid-type labeling is required, the labeled amino acid is added to the fermentation of the expression host (topic 1 above, see Sect. 1.2.3). In this case, a thorough isotope analysis of the expressed protein is advisable prior to NMR spectroscopic investigations. This is preferentially achieved by GC-MS analyses of the hydrolyzed amino acids from the protein product. 

The target macromolecule must first be isotopically labeled either at aU amino acid residues (uniform labeling), at specific types of amino acids (specific labeling), or with C labels scattered across the entire molecule but not covering aU sites. This is readily achieved when the protein is amenable to overexpression in bacterial cells supplemented with appropriate and N labeled growth media. A basic approach is to collect two-dimensional or... 

For the residue-type assignment of the cross peaks, the SH3 domain was expressed with specifically labeled glycine or arginine residues by using amino acid mixtures where only Gly and Arg were 15N-labeled. Again, yields for the labeled proteins were identical to those of the unlabeled product. Analysis of the corresponding [15N,1H]-HSQC spectra... 

While general labeling strategies relying on expression of proteins using hydrolyzates of algae are useful for uniform or amino acid-specific labeling, expression can also be performed in minimal media in a cost-effective manner. These types of expression media... 

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