Post-translational modification states

As described in more detail below, agonist binding will lead to signaling as well as phosphorylation of Ser and Thr residues, especially, but also, in selected cases, Tyr residues located in intracellular loop-3 and in the C-terminal extension. This post-translational modification alters the affinity of the receptor for various intracellular proteins, including arrestin, which sterically prevents further G-protein binding and functions as an adaptor protein. Also, interaction with other types of scaffolding proteins such as PSD-95-like proteins, is influenced by the phosphorylation state of the receptor. 

The formation of an aldehyde group on a macromolecule can produce an extremely useful derivative for subsequent modification or conjugation reactions. In their native state, proteins, peptides, nucleic acids, and oligonucleotides contain no naturally occurring aldehyde residues. There are no aldehydes on amino acid side chains, none introduced by post-translational modifications, and no formyl groups on any of the bases or sugars of DNA and RNA. To create reactive aldehydes at specific locations within these molecules opens the possibility of directing modification reactions toward discrete sites within the macromolecule. 

As stated in the introduction, the large-scale production of recombinant slgA is a very challenging task. This is due to the complex post-translational modifications that are required and because two distinct cell types are needed to produce the native... 

The proteome is the set of expressed proteins at a given time under defined conditions it is dynamic and varies according to the cell type and functional state. One of the main differences when working with proteins is that there is not an amplification methodology for proteins comparable to PCR. Physical and chemical diversity of proteins are also higher than nucleic acids. They differ among individuals, cell types, and within the same cell depending on cell activity and state. In addition, there are hundreds of different types of post-translational modifications (PTMs), which evidently will influence chemical properties and functions of proteins. PTMs are key to the control and... 

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

In the focus of a renewed interest are histone MTases. They produce monomethylarginine, symmetrical and nnsymmetrical dimethylarginine, and all possible lysine e-amino gronp methy-lation states. Such post-translational modifications of the histones determines whether chromatin adopts a compacted structure and is associated with silenced DNA-heterochromatin, or if it seems to be an extended structure and is associated with transcriptionally active DNA-euchromatin (20). Arginine MTases seem to methylate even more substrates, but the modification effects are not well nnderstood (21). As in DNA, the above-described examples of methylated residnes can be viewed as steric marks designed for recognition by highly specific proteins. 

Parallel to the efforts of identifying substrate proteins for a particular enzyme, the activity-based small molecule probes pioneered by Cravatt and coworkers can facilitate the identification of the biological functions of an enzyme that catalyzes protein post-translational modifications (145). The major advantage of this type of probes is that potentially they can detect enzymes that are in the active states, and thus can provide snapshots of enzymes that are in the active states at different development stages or different types of cells. Among enzymes that catalyze PTM, so far probes have been developed for studying proteases (145, 146), kinases (147), pTyr phosphatases (148), and protein Arg deiminases (149). 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

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