Primary structure separated peptides

In biological recognition phenomena, protein-protein interactions are of primary importance. In an attempt to mimic these processes, LaBrenz and Kelly [51] synthesized the peptidic host 64. In this receptor, the dibenzofuran template separates the two peptide units by roughly 10 A and allows for the complexation of a guest peptide (65), as depicted in Fig. 21. The complex first forms a three-stranded, antiparallel /J-sheet that is stabilized by hydrogen bonds, electrostatic interactions, and aromatic-aromatic interactions between the dibenzofuran and the benzamide moieties. This complex can further self associate to form more complex structures. This example shows that structurally defined peptide nanostructures can interfere with biological recognition processes and potentially have therapeutic applications. 

The primary level of structure in a protein is the linear sequence of amino acids as joined together by peptide bonds. This sequence is determined by the sequence of nucleotide bases in the gene encoding the protein (see Topic HI). Also included under primary structure is the location of any other covalent bonds. These are primarily disulfide bonds between cysteine residues that are adjacent in space but not in the linear amino acid sequence. These covalent cross-links between separate polypeptide chains or between different parts of the same chain are formed by the oxidation of the SH groups on cysteine residues that are juxtaposed in space (Fig. 4). The resulting disulfide is called a cystine residue. Disulfide bonds are often present in extracellular proteins, but are rarely found in intracellular proteins. Some proteins, such as collagen, have covalent cross-links formed between the side-chains of Lys residues (see Topic B5). 

Figure 24-10 shows the structure of insulin, a more complex peptide hormone that regulates glucose metabolism. Insulin is composed of two separate peptide chains, the A chain, containing 21 amino acid residues, and the B chain, containing 30. The A and B chains are joined at two positions by disulfide bridges, and the A chain has an additional disulfide bond that holds six amino acid residues in a ring. The C-terminal amino acids of both chains occur as primary amides. 

Following successful recovery of peptide/protein molecule from the microspheres, a simple spectrophotometric method does not always allow discrimination between the monomeric protein form and its aggregates. However, HPLC might separate these species and thus provides more accurate qualitative data [96], But HPLC cannot quantify exclusively the amount of active protein antigen, as is the case with ELISA techniques [97], Nowadays, Fourier transform infrared (FTIR) spectroscopy has become a popular, noninvasive method, as it is able to characterize the secondary structure of entrapped proteins [26, 95, 98-101], Only recently, the integrity of their primary structure was evaluated, thanks to a new matrix-assisted laser... 

The peptides obtained by specific chemical or enzymatic cleavage are separated by some type of chromatography. The sequence of each purified peptide is then determined by the Edman method. At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. How can we order the peptides to obtain the primary structure of the original protein The necessary additional information is obtained from overlap... 

The sequence of amino acids in the polypeptide chain (i.e., the primary structure of a polypeptide or protein) can be established by selective chemical and enzymatic cleavage of the protein followed by separation, amino acid analysis, and sequence determination of all peptide fragments. The entire amino acid sequence is established by overlapping identical regions of the individual fragments. The following problem illustrates the procedure. 

Phenylthiohydantoin derivatization offers a special value because it is actually performed during Edman degradation, the sequencing technique mostly used for the determination of the primary structure of proteins and peptides. PTH derivatives are separated in many different stationary phases, in either normal- or re-versed-phase mode and are mostly detected at 254 nm [8,9]. Using radiolabeled proteins, sequencing of proteins down to the 1-100-pmol range can be achieved. The formed derivatives are basic and thus interact strongly with base silica materials. RP separations are mostly carried out in acidic conditions with the addition of appropriate buffers (sodium acetate mostly, but... 

In 1960, Hirs, Moore, Stein, and Anfinsen described the first primary structure of the enzyme ribonuclease (M.W. 13,700), which has a single peptide chain of 124 amino acid residues and four intrachain disulfide bonds. These investigators established many of the techniques still used in sequence analysis, such as the use of ion exchange resins for separation of peptides and amino acids. 

In contrast to a-helices, /1-sheets do not involve interactions between amino acids close in sequence. Amino acids that interact within / -sheets are often found widely separated in the primary structure. Therefore, / -sheet formation needs structures that bring two polypeptide segments into close proximity. This is achieved via reverse turn structures [96]. Turns are aperiodic or nonrepetitive elements of secondary structure which mediate the folding of the polypeptide chain into a compact tertiary structure. Turns usually occur on the environment-exposed surface of proteins [97,98], Reverse turns play an important role in polypeptide function, both as elements of structure as well as modulators of bioactivity [99]. Among the reverse turns found in proteins the /1-turn is the most relevant [100]. /1-Turns comprise four amino acid residues (i to i+3) forming an almost complete 180° turn in the direction of the peptide chain [101,102]. 

Biochemists distinguish four levels of the structural organization of proteins. In primary structure, the amino acid residues are connected by peptide bonds. The secondary structure of polypeptides is stabilized by hydrogen bonds. Prominent examples of secondary structure are a-helices and / -pleated sheets. Tertiary structure is the unique three-dimensional conformation that a protein assumes because of the interactions between amino acid side chains. Several types of interactions stabilize tertiary structure the hydrophobic effect, electrostatic interactions, hydrogen bonds, and certain covalent bonds. Proteins that consists of several separate polypeptide subunits exhibit quaternary structure. Both noncovalent and covalent bonds hold the subunits together. 

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