Labeling amino-acid type

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. 

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. 

Fig. 4.4 Example of amino acid-type selective labeling. A [ H,15N]-HSQC spectrum obtained with the fully 15N-labeled monomeric form of the KSHV protease. B and C [nH,15N]-l-ISQC 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... 

Instead of using amino acid-type selective labeling suppression of high background signals can also be achieved by selective expression of the protein of interest with the concomitant suppression of the expression of all other (host-) proteins. In principle, this suppression of host gene expres-... 

Another way of overcoming spectral overlap for proteins is selective isotope labeling of one or several amino acid types, which results in less-crowded H- N spectra with cross-peaks from the labeled residues only. The same is possible for RNA where nucleotides can be selectively isotope enriched. In addition, methods for segmental labeling of parts of the biomolecules have been developed for proteins (48, 49) and for RNA (50). 

Give the general formula for an amino acid. Some amino acids are labeled hydrophilic and some are labeled hydrophobic. What do these terms refer to Aqueous solutions of amino acids are buffered solutions. Explain. Most of the amino acids in Fig. 22.18 are optically active. Explain. What is a peptide bond Show how glycine, serine, and alanine react to form a tripeptide. What is a protein, and what are the monomers in proteins Distinguish between the primary, secondary, and tertiary structures of a protein. Give examples of the types of forces that maintain each type of structure. Describe how denaturation affects the function of a protein. 

Fig. 3 Type 11 dehydroquinase a) a ribbon representation of the quaternary structure of the enzyme, with each peptide chain depicted in a different color. The inhibitor 2,3-anhydro-quinic acid is shown in space-filling representation, with atoms colored according to atom type, b) On the left is a detailed view of the enzyme active site as seen in the crystal structure, with the ligand highlighted in green and key amino acid residues labeled. This is compared with the traditional two-dimensional representation of the two steps of the enzyme mechanism on the right. (View this an in color at WWW.dekker.com.)...

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... 

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