N-terminal polypeptide

The role of catalysis in membrane assembly is emphasized again by the above model since the N-terminal sequence of the nascent polypeptide chain of a spanning protein is released by proteolysis as soon as it reaches the cytosol. The N-terminal polypeptide chain extension may help the chain penetrate the hydrophobic bilayer and solubilize the resulting hydrophobic N-terminal part of the chain in the aqueous medium of the cytoplasm. However, the role of the protease-catalyzed hydrolysis of the polypeptide chain in membrane assembly is minimized in the membrane trigger hypothesis (99). According to this model, the essential role of the leader sequence would be to modify, in association with the lipid bilayer, the folding pathway of the protein in such a way that the polypeptide chain could span the membrane. 

The second approach, N-terminal polypeptide D (gD ) trkA KIRA using CHO cells stably transfected with a recombinant human trkA receptor with an gD flag, is developed to measure the bioactivity of NGF. A polypeptide flag is cloned onto the N-terminus or C-terminus of the full-length recombinant human receptor stably transfected into CHO cells. A 26-amino-acid polypeptide derived from HSV gD as a capture reagent in the ELISA phase of the KIRA and a mAb 3C8 antibody as the capture antibody are used. 

The observations that have been summarized immediately suggest structure-function relationship. It seems that a 15-residue segment of the C-terminal polypeptide chain is not essential to activity. Changes in the amino acid sequence can take place without inducing loss of activity. Such a view is supported by degradation experiments. Pepsin degradation of the 39-resi-due polypeptide leads to the formation of three smaller but active polypeptides one of 28, one of 30, and one of 33 amino acid residues. Each of these polypeptides includes the N-terminal serine. In contrast, enzymic (carboxypeptidase, pepsin, chymotrypsin) hydrolysis of the 24 N-terminal polypeptide destroys hormonal activity. Furthermore, the elimination of the single amino acid of the 24 N-terminal peptide seems to inactivate the molecule completely. 

The polypeptide chain of the 92 N-terminal residues is folded into five a helices connected by loop regions (Figure 8.6). Again the helices are not packed against each other in the usual way for a-helical structures. Instead, a helices 2 and 3, residues 33-52, form a helix-turn-helix motif with a very similar structure to that found in Cro. 

The helices at the N-terminal regions of the two polypeptide chains are intertwined and make extensive contacts in the central part of the molecule to form a stable core. This core supports two "heads", each comprising the last three helices from one polypeptide chain. Alpha helix 3 in the middle of the subunit chain is quite long and forms the main link between the core and the head. 

The polypeptide chain of the lac repressor subunit is arranged in four domains (Figure 8.21) an N-terminal DNA-hinding domain with a helix-turn-helix motif, a hinge helix which binds to the minor groove of DNA, a large core domain which binds the corepressor and has a structure very similar to the periplasmic arablnose-binding protein described in Chapter 4, and finally a C-terminal a helix which is involved in tetramerization. This a helix is absent in the PurR subunit structure otherwise their structures are very similar. 

Many biochemical and biophysical studies of CAP-DNA complexes in solution have demonstrated that CAP induces a sharp bend in DNA upon binding. This was confirmed when the group of Thomas Steitz at Yale University determined the crystal structure of cyclic AMP-DNA complex to 3 A resolution. The CAP molecule comprises two identical polypeptide chains of 209 amino acid residues (Figure 8.24). Each chain is folded into two domains that have separate functions (Figure 8.24b). The larger N-terminal domain binds the allosteric effector molecule, cyclic AMP, and provides all the subunit interactions that form the dimer. The C-terminal domain contains the helix-tum-helix motif that binds DNA. 

The polypeptide chain of p53 is divided in three domains, each with its own function (Figure 9.16). Like many other transcription factors, p53 has an N-terminal activation domain followed by a DNA-binding domain, while the C-terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers. Mutants lacking the C-terminal domain do not form tetramers, but the monomeric mutant molecules retain their sequence-specific DNA-binding properties in vitro. 

The 12 residues between the second cysteine zinc ligand and the first histidine ligand of the classic zinc finger motif form the "finger region". Structurally, this region comprises the second p strand, the N-terminal half of the helix and the two residues that form the turn between the p strand and the helix. This is the region of the polypeptide chain that forms the main interaction area with DNA and these interactions are both sequence specific. 

The C-terminal transmembrane helix, the inner helix, faces the central pore while the N-terminal helix, the outer helix, faces the lipid membrane. The four inner helices of the molecule are tilted and kinked so that the subunits open like petals of a flower towards the outside of the cell (Figure 12.10). The open petals house the region of the polypeptide chain between the two transmembrane helices. This segment of about 30 residues contains an additional helix, the pore helix, and loop regions which form the outer part of the ion channel. One of these loop regions with its counterparts from the three other subunits forms the narrow selectivity filter that is responsible for ion selectivity. The central and inner parts of the ion channel are lined by residues from the four inner helices. 

The phosducin polypeptide chmn, of some 240 amino acids, is folded into two domains (Figure 13.16). The N-terminal domain is mostly a-helical and appears to be quite flexible since only a weak electron density is obtained in the structure determination. The actual path of the polypeptide chain from the end of helix to the beginning of helix Ba is tentative due to slight disorder. This region is close to serine 73 at the beginning of Ba, which also becomes disordered on phosphorylation. 

The polypeptide chain of Src tyrosine kinase, and related family members, comprises an N-terminal "unique" region, which directs membrane association and other as yet unknown functions, followed by a SH3 domain, a SH2 domain, and the two lobes of the protein kinase. Members of this family can be phosphorylated at two important tyrosine residues—one in the "activation loop" of the kinase domain (Tyr 419 in c-Src), the other in a short... 

The N-terminal part of the tomato bushy stunt virus polypeptide chain (the R-segment in Figure 16.8) is disordered in all the subunits. As in the core of many other single-strand RNA viruses this region of the polypeptide chain... 

Figure 17.3 The polypeptide chain of lysozyme fiom hacteiiophage T4 folds into two domains. The N-terminal domain is of the a + P type, built up from two a helices (red) and a four-stranded antiparallel P sheet (green). The C-terminal domain comprises seven short a helices (brown and blue) in a rather irregular arrangement. (The last half of this domain is colored blue for clarity.)...

Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but it does give amounts from which the percentages or ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-termuial end. 

The unique characteristic of each protein is the distinctive sequence of amino acid residues in its polypeptide chain(s). Indeed, it is the amino acid sequence of proteins that is encoded by the nucleotide sequence of DNA. This amino acid sequence, then, is a form of genetic information. By convention, the amino acid sequence is read from the N-terminal end of the polypeptide chain through to the C-terminal end. As an example, every molecule of ribonucle-... 

After mRNA splicing, the tropoelastin mRNA is translated at the surface of the rough endoplasmic reticulum (RER) in a variety of cells smooth muscle cells, endothelial and microvascular cells, chondrocytes and fibroblasts. The approximately 70 kDa precursor protein (depending on isoform) is synthesized with an N-terminal 26-amino-acid signal peptide. This nascent polypeptide chain is transported into the lumen of the RER, where the signal peptide is removed cotranslationally [9]. 

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