C-terminal residues

The C-terminal residue is determined by the use of either a chemical reagent or the enzyme carboxypeptidase. The 

Phenylthiohydantoin derivative of N-terminal amino acid (PTH-amino acid) 

Determination of the N-terminal residue by the Edman procedure. After removal of the N-terminal amino acid, the remainder of the peptide remains intact and a new N-terminal amino acid is available for removal by the next reaction cycle. 

Carboxypeptidase is an exopeptidase that specifically hydrolyzes the C-terminal peptide bond and releases the C-terminal amino acid. Two problems are associated with its use the substrate specificity of the enzyme and the continuous action of the enzyme. The continuous action may yield the second, third, and additional residues from some chains even before the terminal residues on every chain are quantitatively released. Thus, it may be difficult to determine which residue is the C terminus. However, monitoring the sequential release of amino acids can often reveal the sequence of several residues at the C terminus. Concerning specificity, carboxypeptidase A releases all C-terminal residues except Lys, Arg, and Pro carboxypeptidase B cleaves C-terminal Arg and Lys residues and carboxypeptidase C hydrolyzes C-terminal Pro residues. Thus, more than one method may be needed to establish the C-terminal amino acid. 

Numerous methods were proposed for this purpose, but only few withstood the test of time. A reliable procedure is hydrazinolysis (Akabori et al. 1952) which involves the heating of a solution of the peptide in ca. 97 % hydrazine in a sealed tube at 100 °C for 12 hours. The peptide bonds are cleaved by hydrazine and the amino acid constituents thus converted to amino acid hydrazides except the C-terminal residue which is merely hberated in the process. Its separation is faciUtated by dinitrophenylation of the mixture with 2,4-dinitrofluorobenzene. The DNP-amino acid hydrazides as neutral substances are extracted from the aqueous, bicarbonate containing mixture with an organic solvent while the sodium salt of the DNP-derivative of the C-terminal amino acid remains dissolved  

Of course, the DNP-derivatives of the dicarboxylic acid monohydrazides also remain in the aqueous layer. 

Subtractive methods have been applied for the determination of the C-terminal residue in various ways. For instance, treatment of a peptide with a mixture of acetic anhydride and pyridine at elevated temperature (Dakin and 

West 1928) converts the C-terminal residue to a methyl keton, presumably through a mixed anhydride and an azlactone 

Hydrolysis of samples of both starting material and product followed by amino acid analysis of the hydrolysates reveals the C-terminal residue by its absence from the hydrolysate of the treated peptide. In an analogous manner the C-terminal residue can be transformed to a non-amino acid moiety by reduction with LiAlH4 or LiBH4 (Fromageot et al. 1950). More reliable results are obtained if prior to reduction the carboxyl group is esterified with the help of diazomethane. In addition to subtraction the C-terminal residue can be identified in the form of the resulting aminoalcohol as well  

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Figure 8.3 The DNA-binding protein Cro from bacteriophage lambda contains 66 amino acid residues that fold into three a helices and three P strands, (a) A plot of the Ca positions of the first 62 residues of the polypeptide chain. The four C-terminal residues are not visible in the electron density map. (b) A schematic diagram of the subunit structure. a helices 2 and 3 that form the helix-turn-helix motif ate colored blue and red, respectively. The view is different from that in (a), [(a) Adapted from W.F. Anderson et al., Nature 290 754-758, 1981. (b) Adapted from D. Ohlendorf et al., /. Mol. Biol. 169 757-769, 1983.]...

The side of the p sheet that faces away from DNA is covered by two long a helices. One of these helices contains a number of basic residues from the middle segment of the polypeptide chain while the second helix is formed by the C-terminal residues. Residues from these two helices and from the short loop that joins the two motifs (red in Figure 9.4) are likely candidates for interactions with other subunits of the TFIID complex, and with specific transcription factors. 

For the identification of the C-terminal residue of polypeptides, an enzymatic approach is commonly used. 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

FIGURE 9.19 Proteins containing the C-terminal sequence CAAX can undergo prenylation reactions that place thioether-linked farnesyl or geranylgeranyl groups at the cysteine side chain. Prenylation is accompanied by removal of the AAX peptide and methylation of the carboxyl group of the cysteine residue, which has become the C-terminal residue. 

FIGURE 7.7 C-terminal residues of G-protein a subunits. The cysteine ADP-ribosylated by Pertussis toxin (PTx) is boxed. 

Figure 10-5. Representative conformations of the (5 amyloid peptide (10-42) under different pH conditions. The conformations were obtained as centroids of the most populated clusters from the replica-exchange CPHMD folding simulations [43, 44]. The N-terminal residues 10-28 are shown in blue the C-terminal residues 29-42 are shown in red. In the most aggregation-prone state (pH 6), the side chains of the central hydrophobic cluster Leu-17, Val-18, Phe-19, Phe-20 and Ala-21 are shown as van der Waals spheres in pink, grey, cyan, purple and green, respectively...

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... 

Attachment of the C-Terminal Residue Through its Amino Group Via On-Resin Reductive Amination... 

With the C-terminal residue introduced as part of the BAL anchor and the penultimate residue incorporated successfully by the optimized acylation conditions just described, further stepwise chain elongation by addition of Fmoc-amino acids generally proceeded normally by any of a variety of peptide synthesis protocols. 

Figure 10.10 Amino acid sequence of human adrenocorticotrophin. The amino acid residues in this polypeptide hormone are linked by peptide bonds and each residue is given a number starting with the N-terminal residue (number 1) to the C-terminal residue (number 39).

There is a report that the signal for the outer membrane assembly resizes at the C termini of proteins (Struyve et al., 1991 de Cock et al., 1997). That is, most C-terminal residue must be phenylalanine in the outer membrane proteins. Experiments using mutant proteins showed that C-terminal phenylalanine is important for the efficient assembly of PhoE. 

Further susceptibilities of the nonapeptide are shown in Fig. 6.34. Thus, the Pro7-Phe8 bond is also cleaved by neprilysin (EC 3.4.24.11) and endothelin-converting enzyme (EC 3.4.24.71) [149][165]. The C-terminal residue is susceptible to cleavage by several carboxypeptidases, particularly carboxypeptidase N. Thimet oligopeptidase (EC 3.4.24.15) is also able to act on bradykinin, which is cleaved at the Phe5-Ser6 bond [144],... 

Figure 10.10 Stability studies analysis by LC-MS. Long-term stability studies (3 months, 30°C) are evaluated by LC-MS analysis of a C-terminal peptide fragment. Various degradation mechanisms are visualized, removal of C-terminal residues due to proteolytic activities, isomerization and deamidation of specific asparagine residues. Future development efforts will allow the use of this methodology to assess progress toward a stable formulation.

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