Modification with myristoyl

The increase in the length of the side chain results normally in an internal plasticization effect caused by a lower polarity of the main chain and an increase in the configurational entropy. Both effects result in a lower activation energy of segmental motion and consequently a lower glass transition temperature. The modification of PPO with myristoyl chloride offers the best example. No side chain crystallization was detected by DSC for these polymers. 

Proteins with an isoprenoid modification possess either a C15-famesyl residue or a C20-geranyl-geranyl residue. Both residues are bound via a thioester linkage to a cysteine residue. As with myristoylation, these are constitutive, stable modification performed by farnesyl or geranyl transferases. 

Another wide application of mass spectrometry is the detection and characterization of post-translational modifications such as myristoylation, phosphorylation, disulfide bridging, etc. The detection and localization of post-transla-tional modifications has been a rapidly developing area of mass spectrometry due to the functional importance of these modifications in biological systems. An example of this was recently shown for the case of the human rhinovirus HRV14 [10]. Electron density maps from crystallography data indicated a myristoylation of VP4. Mass analysis of VP4 also indicated a mass difference of 212 Da (consistent with myristoylation of VP4). Additional experiments with proteolytic digestion and tandem mass spectrometry were able to localize the modification to the N-terminus of VP4. 

Some proteins can be posttranslationally modified by the addition of prenyl groups. Prenyl groups are long-chain, unsaturated hydrocarbons that are intermediates in isoprenoid synthesis. The farnesyl group has 15 carbons, and the geranylgeranyl has 20 carbons. They are attached to a cysteine residue near the end of the protein as a thiol ether (Protein-S-R). Other proteins can have a long-chain fatty acid (C14=myristoyl, C16=palmitoyl) attached to the amino terminus as an amide. These fatty acid modifications can increase the association of proteins with the membrane. 

N-Myristoylation is achieved by the covalent attachment of the 14-carbon saturated myristic acid (C14 0) to the N-terminal glycine residue of various proteins with formation of an irreversible amide bond (Table l). 10 This process is cotranslational and is catalyzed by a monomeric enzyme called jV-myri s toy 11ransferase. 24 Several proteins of diverse families, including tyrosine kinases of the Src family, the alanine-rich C kinase substrate (MARKS), the HIV Nef phosphoprotein, and the a-subunit of heterotrimeric G protein, carry a myr-istoylated N-terminal glycine residue which in some cases is in close proximity to a site that can be S-acylated with a fatty acid. Functional studies of these proteins have shown an important structural role for the myristoyl chain not only in terms of enhanced membrane affinity of the proteins, but also of stabilization of their three-dimensional structure in the cytosolic form. Once exposed, the myristoyl chain promotes membrane association of the protein. 5 The myristoyl moiety however, is not sufficiently hydrophobic to anchor the protein to the membrane permanently, 25,26 and in vivo this interaction is further modulated by a variety of switches that operate through covalent or noncovalent modifications of the protein. 4,5,27 In MARKS, for example, multiple phosphorylation of a positively charged domain moves the protein back to the cytosolic compartment due to the mutated electrostatic properties of the protein, a so-called myristoyl-electrostatic switch. 28 ... 

As discussed for N-myristoylation and S-prenylation, even S-acylation of proteins with a fatty acid which in the vast majority of cases is the C16 0 palmitic acid, plays a fundamental role in the cellular signal-transduction process (Table l). 2-5 14 While N-myristoylation and S-prenylation are permanent protein modifications due to the amide- and sulfide-type linkage, the thioester bond between palmitic acid and the peptide chain is rather labile and palmi-toylation is referred to as a dynamic modification. 64 This reversibility plays a crucial role in the modulation of protein functions since the presence or absence of a palmitoyl chain can determine the membrane localization of the protein and can also be used to regulate the interactions of these proteins with other proteins. Furthermore, a unique consensus sequence for protein palmitoylation has not been found, in contrast to the strict consensus sequences required for N-myristoylation and S-prenylation. Palmitoylation can occur at N- or C-terminal parts of the polypeptide chain depending on the protein family and often coexists with other types of lipidation (see Section 6.4.1.4). Given the diversity of protein sequences... 

Palmitoylation is, after myristoylation, the most common modification of the a-sub-rmit of the heterotrimeric G-proteins (see chapter 5). The a-subunit of G-proteins can be lipidated in a two-fold marmer, with a myristoic acid and a pahnitoic acid anchor at the N-terminus. It appears in this case that two lipid anchors are necessary to mediate a stable association of the protein with the membrane. The lipidation of cytoplasmic protein tyrosine kinase also includes both myristoylation and palmitoylation. H-Ras protein also requires, apart from C-terminal farnesylation (see below), a pahnitoyl modification in order to bind to the plasma membrane. In all mentioned examples the fatty acid anchors play an essential role in the signal transduction. 

The function of many adaptor proteins is closely linked with the cell membrane or with the cytoskeleton. The occurrence of PH domains and myristoyl modifications suggests that adaptor proteins are involved in particular in coordination and assembly of signal complexes on the iimer side of the ceU membrane. 

Data from in vitro activity assays with these purified recombinant proteins can typically be interpreted much more easily than data obtained from experiments with crude or partially purified protein extracts, because (1) there will be no competing proteins with similar activity present in the assay, and (2) there will no enzymes present that convert the product generated by the enzyme of interest, and hence reduce the effective product concentration. A potential downside of the use of recombinant protein over crude extracts is the fact that critical co-factors that will ensure proper activity may not be present in the purified protein fraction. If that is the case, the researcher will have to empirically determine which co-factor and at what concentration needs to be included in the assay. Another consideration is that the native protein may have undergone post-translational processing, such as acetylation, glycosylation, myristoylation, etc. These modifications may not occur or may not occur properly when the protein is expressed in bacterial, fungal or insect cells. Assuming that these potential problems do not occur or can be dealt with, the availability of pure recombinant protein will enable the determination of substrate specificity, as well as kinetic experiments in which the rate of conversion is measured as a function of time and/or substrate concentration. 

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

In addition to creating recognition motifs to recruit proteins, a few PTM can also increase interaction with other species, such as the lipid bilayer of different cellular membranes. These modifications include the formation of GPI-anchored proteins (20), protein myristoylation on the a-amino group of the N-terminal... 

Fusion protein of AKT with an amino-terminal myristoylation signal sequence. This fatty acid modification promotes persistent plasma membrane association and signaling activity. 

Thioacylated proteins contain fatty acids in thioester linkage to cysteine residues [7-9] (Fig. lA). Protein thioacylation is frequently referred to as palmitoylation, although fatty acids other than palmitate are found on thioacylated proteins. Membrane proteins as well as hydrophilic proteins are thioacylated, the latter, in many cases, acquiring the modification when they become associated with a membrane compartment as a result of initial N-myristoylation or prenylation. Examples include G-protein coupled receptors, the transferrin receptor, the cation-dependent mannose-6-phosphate receptor, and hydrophilic proteins such as members of the Src family of protein tyrosine kinases (e.g., p59h " and p56 ) as well as H-Ras, N-Ras, and the synaptic vesicle protein SNAP-25. The yeast palmitoyl proteome, i.e., the collection of all S-acylated proteins in yeast, was recently defined via a comprehensive proteomics approach (A.F. Roth, 2006). It consists of 50 proteins including... 

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