N-terminal glycines

Myristic acid may be linked via an amide bond to the a-amino group of the N-terminal glycine residue of selected proteins (Figure 9.18). The reaction is referred to as A -myristoylation and is catalyzed by myristoyl—CoAtprolein N-myris-toyltransferase, known simply as NMT. A -Myristoyl-anchored proteins include the catalytic subunit of cAMP-dependent protein kinase, the ppSff tyrosine kinase, the phosphatase known as calcineurin B, the a-subunit of G proteins (involved in GTP-dependent transmembrane signaling events), and the gag proteins of certain retroviruses, including the FHV-l virus that causes AIDS. 

Myristoylation is the post-translational addition of the 14-carbon fatty acid myristate to the N-terminal glycine of proteins via an amide link. Myristoylation of proteins helps to anchor them to membranes. 

Chemical shift in acidic media is defined as at pH <2.0. Chemical shift in basic media is defined as at pH >11.0 for more details, see Ref. 23. b C of N-terminal glycine exhibited pH-dependence at pH >7.0. C of C-terminal glycine exhibited pH-dependence at pH <7.0. 

Silk fibroin contains no cystine and the content of lysine and histidine is also low (about 1% in total), but it does contain tyrosine phenolic (13%) and serine alcoholic (16%) sidechains. Since glycine accounts for 44% of the total aminoacid content, an N-terminal glycine residue is reasonably representative of most of the primary amino dyeing sites in silk fibres. Amino acid analysis of hydrolysed reactive-dyed silk indicates that the reaction between fibroin and reactive dyes takes place mainly at the e-amino group of lysine, the imino group of histidine and the N-terminal amino group of the peptide chain. In an alkaline medium, the hydroxy groups of tyrosine and serine also react [114]. 

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 ... 

Cys-Tyr-Ile-Gln-AsivCys-Pro-Arg-GlyNH2 N terminal Glycine amide, C-terminal... 

An example is provided by actin, which contains acetyl-Met-Asp, acetyl-Met-Gln or acetyl-Met-Cys-Asp at the N terminus immediately after synthesis. Then, within 15 min the acetyl-Met is cleaved off, and the next terminal residue is acetylated.548 N-Acylation of nascent peptides by fatty acyl groups can also occur cotranslationally. For example, 14-carbon myristoyl groups are added in amide linkage to the N-terminal glycines of many cellular and virally encoded proteins.535 549 550 This may take place on the ribosomes,551... 

In a more elaborate example, Thamm et al. (1980) selectively removed, by Edman degradation, the N-terminal glycine of the A-chain of an insulin derivative in which the B-chain amino terminus and the B-chain lysine were protected. The new N-terminus was reacted with theN-hydroxysuc-cinimide ester of A-(4-azido-2-nitrophenyl)glycine to yield, after deprotection, a derivative of insulin modified only at the NaA-terminus. The amino protecting group chosen for this procedure was the base labile, methylsulfonylethyloxycarbonyl. 

The overmodification of collagen from patients 1 and 2 is weak because it occurs only on the N-terminal side of the mutation. Mutations located closer to the C-terminal end lead to substantially larger overhydroxylation and overgly-cosylation. In principle, this overmodification gradient could explain milder OI symptoms observed for N-terminal glycine substitutions (e.g., almost no lethal substitutions in the N-ter-minal quarter of collagen were reported, and many of these cases were classified as type I OI). However, no consistent severity gradient and no correlations of symptoms with over-modification were found for mutations located further toward the C-terminal end. Thus, the role of overmodification in OI phenotype still remains unclear. 

Acyltransferases are also known that catalyze acyl transfer from longer chain acyl-CoA. The highly stndied protein N-myristoyltransferase catalyzes transfer of the myristoyl gronp to the amine gronp of N-terminal glycine of a protein snbstrate... 

Gray C, Tatulian SA, Wharton SA, Tamm LK. Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers. Biophys. J. 1996 70 2275-2286. 

G Protein is a class of proteins, occurring at the plasma membrane, that participates in transmitting signals from the extracellular fluid to various enzymes within the cell. Myristic acid is bound to G protein. Specifically, myristic acid is attached to the a subunit of G protein. This lipid is bound via an amide linkage to the N-terminal glycine. A geranylgeranyl group is attached via a thiol-ester bond to a cysteine residue on the y subunit of G protein (Casey and Seabra, 1996). 

A group of cytosolic proteins are anchored to the cytosolic face of a membrane by a fatty acyl group (e.g., myristate or palmitate) attached to the N-terminal glycine residue (Figure 5-15a). Retention of such proteins at the membrane by the N-terminal acyl anchor may play an important role in a membrane-associated function. For example, v-Src, a mutant form of a cellular tyrosine kinase, is oncogenic and can transform cells only when it has a myristylated N-terminus. 

Myristoylation is typically considered to be an early co-translational event that occurs in the cytoplasm as soon as -60 amino acids of the nascent peptide emerge from the ribo-somal tunnel (I. Deichaite, 1988), and after the N-terminal glycine residue is made available by cellular methionyl-aminopeptidases that remove the initiator methionine residue. However, myristate can be attached post-translationally to N-terminal glycine in synthetic peptides of the appropriate sequence, and also to N-terminal glycines that are newly generated after caspase-mediated cleavage of proteins during apoptotic cell death. The latter process is termed morbid myristoylation (J. Zha, 2000 M. Mishkind, 2001). 

Acylation - transfer of single saturated acyl groups. These are primarily myristate (C14) linked as an amide to N-terminal glycine and palmitate (C16) linked as a thioester to cysteine. A large number of viral proteins, membrane proteins, and proteins involved in signal transduction are acylated in this manner. 

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