Amino-acid-type-selective labeling

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...

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-... 

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. 

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). 

The 140-residue protein AS is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson s disease. Full-length 13C/15N-labelled AS fibrils and AS reverse-labelled for two of the most abundant amino acids, K and V, were examined by homonuclear and heteronuclear 2D and 3D NMR.147 Two different types of fibrils display chemical shift differences of up to 13 ppm in the l5N dimension and up to 5 ppm for the backbone and side-chain 13C chemical shifts. Selection of regions with different mobility indicates the existence of monomers in the sample and allows the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues are mobile and lack a defined secondary structure, whereas the N terminus is rigid starting from residue 22. In addition, temperature-dependent sensitivity enhancement is also noted for the AS fibrils due to both the CP efficiency and motional interference with proton decoupling.148... 

In a recent study, a serine-phospholipid-selective phospholipase A has been purified and cloned its cDNA from rat platelets, which secrete two types of phospholipases upon stimulation (71). The purified enzyme from extracellular medium of activated rat platelets, yielded a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. The presence of active serine residues was confirmed by labeling the 55-kDa protein with pHJDiisopropyl fluorophosphate, an inhibitor of the enzyme. Based on cDNA for the enzyme cloned from a rat megakaryocyte cDNA library, the 456-amino acid... 

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