A-Polypeptides

As more protein structures became available it was observed that some contained more that one distinct region, with each region often having a separate function. Each of these region is usually known as a domain, a domain being defined as a polypeptide chain that can folc independently into a stable three-dimensional structure. 

Domain Sequence of a polypeptide chain that can independently fold into a stable three-dimensional structure... 

Transfer RNA (tRNA) Transfer RNAs are relatively small nucleic acids containing only about 70 nucleotides They get their name because they transfer ammo acids to the ribosome for incorporation into a polypeptide Although 20 ammo acids need to be transferred there are 50-60 tRNAs some of which transfer the same ammo acids Figure 28 11 shows the structure of phenylalanine tRNA (tRNA ) Like all tRNAs it IS composed of a single strand with a characteristic shape that results from the presence of paired bases m some regions and their absence m others... 

Biosynthetic Human Insulin from E. coli. Insulin [9004-10-8] a polypeptide hormone, stimulates anaboHc reactions for carbohydrates, proteins, and fats thereby producing a lowered blood glucose level. Porcine insulin [12584-58-6] and bovine insulin [11070-73-8] were used to treat diabetes prior to the availabiHty of human insulin [11061 -68-0]. AH three insulins are similar in amino acid sequence. EH LiHy s human insulin was approved for testing in humans in 1980 by the U.S. EDA and was placed on the market by 1982 (11,12). 

For example, a polypeptide is synthesized as a linear polymer derived from the 20 natural amino acids by translation of a nucleotide sequence present in a messenger RNA (mRNA). The mature protein exists as a weU-defined three-dimensional stmcture. The information necessary to specify the final (tertiary) stmcture of the protein is present in the molecule itself, in the form of the specific sequence of amino acids that form the protein (57). This information is used in the form of myriad noncovalent interactions (such as those in Table 1) that first form relatively simple local stmctural motifs (helix... 

Nontraditional Hormones. Novel hormones identified ia cardiovascular tissue have profound effects on maintenance of blood pressure and blood volume ia mammals. Atrial natriuretic hormone (ANH) is a polypeptide hormone secreted from the atria of the heart. When the cardiac atrium is stretched by increased blood volume, secretion of ANH is stimulated ANH ia turn increases salt and water excretion and reduces blood pressure (6). Endothelin is a polypeptide hormone secreted by endothehal cells throughout the vasculature. Although endothelin is released into the circulation, it acts locally in a paracrine fashion to constrict adjacent vascular smooth muscle and increase blood pressure (7). 

Factors controlling calcium homeostasis are calcitonin, parathyroid hormone(PTH), and a vitamin D metabolite. Calcitonin, a polypeptide of 32 amino acid residues, mol wt - SGOO, is synthesized by the thyroid gland. Release is stimulated by small increases in blood Ca " concentration. The sites of action of calcitonin are the bones and kidneys. Calcitonin increases bone calcification, thereby inhibiting resorption. In the kidney, it inhibits Ca " reabsorption and increases Ca " excretion in urine. Calcitonin operates via a cyclic adenosine monophosphate (cAMP) mechanism. 

Parathyroid hormone, a polypeptide of 83 amino acid residues, mol wt 9500, is produced by the parathyroid glands. Release of PTH is activated by a decrease of blood Ca " to below normal levels. PTH increases blood Ca " concentration by increasing resorption of bone, renal reabsorption of calcium, and absorption of calcium from the intestine. A cAMP mechanism is also involved in the action of PTH. Parathyroid hormone induces formation of 1-hydroxylase in the kidney, requited in formation of the active metabolite of vitamin D (see Vitamins, vitamin d). 

Much of protein engineering concerns attempts to explore the relationship between protein stmcture and function. Proteins are polymers of amino acids (qv), which have general stmcture +H3N—CHR—COO , where R, the amino acid side chain, determines the unique identity and hence the stmcture and reactivity of the amino acid (Fig. 1, Table 1). Formation of a polypeptide or protein from the constituent amino acids involves the condensation of the amino-nitrogen of one residue to the carboxylate-carbon of another residue to form an amide, also called peptide, bond and water. The linear order in which amino acids are linked in the protein is called the primary stmcture of the protein or, more commonly, the amino acid sequence. Only 20 amino acid stmctures are used commonly in the cellular biosynthesis of proteins (qv). 

The main role of the human thyroid gland is production of thyroid hormones (iodinated amino acids), essential for adequate growth, development, and energy metaboHsm (1 6). Thyroid underfunction is an occurrence that can be treated successfully with thyroid preparations. In addition, the thyroid secretes calcitonin (also known as thyrocalcitonin), a polypeptide that lowers excessively high calcium blood levels. Thyroid hyperfunction, another important clinical entity, can be corrected by treatment with a variety of substances known as antithyroid dmgs. 

Development of a peptide vaccine is derived from the identification of the immunodominant epitope of an antigen (141). A polypeptide based on the amino acid sequence of the epitope can then be synthesized. Preparation of a peptide vaccine has the advantage of allowing for large-scale production of a vaccine at relatively low cost. It also allows for selecting the appropriate T- or B-ceU epitopes to be included in the vaccine, which may be advantageous in some cases. Several vaccines based on peptide approaches, such as SPf66 (95) for malaria and an HIV-1 peptide (142) have been in clinical trials. No peptide vaccines are Hcensed as yet. 

Himdin [8001-27-2] is a polypeptide of 66 amino acids found ia the saUvary gland secretions of the leech Himdo medicinalis (45). It is a potent inhibitor of thrombin and biads to y-thrombia with a dissociation constant of 0.8 x 10 ° M to 2.0 x lO " M. Himdin forms a stable noncovalent complex with free and bound thrombin completely iadependent of AT-III. This material has now been cloned and expressed ia yeast cells (46,47). Its antigenic poteatial ia humans remains to be estabUshed. 

Three hormones regulate turnover of calcium in the body (22). 1,25-Dihydroxycholecalciferol is a steroid derivative made by the combined action of the skin, Hver, and kidneys, or furnished by dietary factors with vitamin D activity. The apparent action of this compound is to promote the transcription of genes for proteins that faciUtate transport of calcium and phosphate ions through the plasma membrane. Parathormone (PTH) is a polypeptide hormone secreted by the parathyroid gland, in response to a fall in extracellular Ca(Il). It acts on bones and kidneys in concert with 1,25-dihydroxycholecalciferol to stimulate resorption of bone and reabsorption of calcium from the glomerular filtrate. Calcitonin, the third hormone, is a polypeptide secreted by the thyroid gland in response to a rise in blood Ca(Il) concentration. Its production leads to an increase in bone deposition, increased loss of calcium and phosphate in the urine, and inhibition of the synthesis of 1,25-dihydroxycholecalciferol. 

A number of different types of ANF have been described, but the original molecule is a polypeptide of 28 amino acids (qv). The prepro-form is released from the atria by stretch, suggesting that increased blood volume in a poody contracting atria compromised by CHF could be one... 

Until the early 1960s, laboratory iavestigators rehed on dialysis for the separation, concentration, and purification of a wide variety of biologic fluids. Examples iaclude removal of a buffer from a proteia solution or concentrating a polypeptide with hyperosmotic dialysate. Speciali2ed fixtures were sometimes employed alternatively, dialysis tubes, ie, cylinders of membrane about the si2e of a test tube and sealed at both ends, were simply suspended ia a dialysate bath. In recent years, dialysis as a laboratory operation has been replaced largely by ultrafiltration and diafiltration. 

Growing ot a polypeptide chain by attachment ot the first aminoadd to an insoluble polymer and attaching successively amirx>acids At the end cleavage of the peptide from the polymer... 

Figure 1.2 Proteins are built up by amino acids that are linked by peptide bonds to form a polypeptide chain, (a) Schematic diagram of an amino acid. Illustrating the nomenclature used in this book. A central carbon atom (Ca) is attached to an amino group (NH2), a carboxyl group (COOH), a hydrogen atom (H), and a side chain (R). (b) In a polypeptide chain the carboxyl group of amino acid n has formed a peptide bond, C-N, to the amino group of amino acid + 1. One water molecule is eliminated in this process. The repeating units, which are called residues, are divided into main-chain atoms and side chains. The main-chain part, which is identical in all residues, contains a central Ca atom attached to an NH group, a C =0 group, and an H atom. The side chain R, which is different for different residues, is bound to the Ca atom.

Figure 1.2 shows one way of dividing a polypeptide chain, the biochemist s way. There is, however, a different way to divide the main chain into repeating units that is preferable when we want to describe the structural properties of proteins. For this purpose it is more useful to divide the polypeptide chain into peptide units that go from one Ca atom to the next Ca atom (see Figure 1.5). Each C atom, except the first and the last, thus belongs to two such units. The reason for dividing the chain in this way is that all the atoms in such a unit are fixed in a plane with the bond lengths and bond angles very nearly the same in all units in all proteins. Note that the peptide units of the main chain do not involve the different side chains (Figure 1.5). We will use both of these alternative descriptions of polypeptide chains—the biochemical and the structural—and discuss proteins in terms of the sequence of different amino acids and the sequence of planar peptide units.

Figure 1.6 Diagram showing a polypeptide chain where the main-chain atoms are represented as rigid peptide units, linked through the atoms. Each unit has two degrees of freedom it can rotate around two bonds, its Ca-C bond and its N-Ca bond. The angle of rotation around the N-Ca bond is called phi (cj)) and that around the Co-C bond is called psi (xj/). The conformation of the main-chain atoms is therefore determined by the values of these two angles for each amino acid.

The fundamental unit of tertiary structure is the domain. A domain is defined as a polypeptide chain or a part of a polypeptide chain that can fold independently into a stable tertiary structure. Domains are also units of function. Often, the different domains of a protein are associated with different functions. For example, in the lambda repressor protein, discussed in Chapter 8, one domain at the N-terminus of the polypeptide chain binds DNA, while a second domain at the C-terminus contains a site necessary for the dimerization of two polypeptide chains to form the dimeric repressor molecule. 

Domains are formed by different combinations of secondary structure elements and motifs. The a helices and p strands of the motifs are adjacent to each other in the three-dimensional structure and connected by loop regions. Sequentially adjacent motifs, or motifs that are formed from consecutive regions of the primary structure of a polypeptide chain, are usually close together in the three-dimensional structure (Figure 2.20). Thus to a first approximation a polypeptide chain can be considered as a sequential arrangement of these simple motifs. The number of such combinations found in proteins is limited, and some combinations seem to be structurally favored. Thus similar domain structures frequently occur in different proteins with different functions and with completely different amino acid sequences. 

The reverse turn as a polypeptide conformation in globular proteins. Proc. Natl. Acad. Sci. USA 70 538-542, 1973. 

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