Examples of Polypeptide and Protein Primary Structure

Using the techniques we described, chemists have had remarkable success in determining the primary structures of polypeptides and proteins. The compounds described in the following pages are important examples. 

When the number of amino acids in a polypeptide chain reaches more than fifty, a protein exists. The structure of both polypeptides and proteins dictate how these biomolecules function. There are several levels of structure associated with polypeptides and proteins. The sequence of the amino acids forming the backbone of the protein is referred to as the primary structure. A different order or even a minor change in an amino acid sequence creates an entirely different molecule. Just reversing the order of amino acids in a dipeptide changes how the dipeptide functions. An example of this is sickle-cell anemia. Sickle-cell anemia is a genetic disorder that occurs when the amino acid valine replaces... 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

Synthetic polypeptides consist of a repeating sequence of certain amino acids and their primary structures are not as complicated as those in proteins. The polypeptides are very important polymers in both polymer and protein science. The characteristic properties related to the structure lead to possible expansion for research in the field of polymer science, to provide very different moplecules from conventional synthetic polymers. For example, the concept of the liquid crystal is expanded by revealing the variety of structures and properties of liquid crystals. Furthermore, the polypeptides are sometimes used as biomimic materials. On the other hand, synthetic polypeptides are sometimes used as model biomolecules for proteins because they take the a-helix, /3-sheet, o)-helix structure, and so on, under appropriate conditions. From such situations, it can be said that synthetic polypeptides are interdisplinary macromolecules and are very important for research work in both polymer and protein science. 

In all, at least 100 different molecules are involved in protein synthesis. Among the most important of these are the components of the ribosome, a supramolec-ular structure composed of RNA and protein that rapidly and precisely decodes genetic messages. Speed is required because organisms must respond expeditiously to ever-changing environmental conditions. In prokaryotes such as E. coli, for example, a polypeptide of 100 residues is synthesized in about 6 s. Precision in mRNA translation is critical because, as described previously, the accurate folding, and therefore the proper functioning, of each polypeptide is determined by the molecule s primary sequence. 

Proteins are biological macromolecules synthesized in cells for specific fiuictions. They are high-molecular-weight polyamides that adopt exquisitely complex structures. This complexity is characterized by different levels of structure primary, secondary, tertiary, and quaternary. Primary structure [6] refers to the amino acid sequence itself, along with the location of disulfide bonds (i.e., covalent connections between two amino acid residues within the protein molecule). Secondary structure refers to the spatial arrangement of amino acid residues that are near one another in the linear sequence. Alpha (a) hehces and beta (fi) sheets are typical examples of a secondary structure. The tertiary structure refers to the spatial arrangement of amino acid residues that are far apart in the linear sequence. If a protein has two or more polypeptide chains, each with its exclusive primary, secondary, and tertiary structure, such chains can associate to form a multichain quaternary structure. Hence, a quaternary structure refers to the spatial arrangement of such subunits and their interaction. 

Biological function in polypeptides and proteins requires a specific three-dimensional shape and arrangement of functional groups, which, in turn, necessitate a definite amino acid sequence. One monkey wrench residue in an otherwise normal protein can completely alter its behavior. For example, sickle-ceU anemia, a potentially lethal condition, is the result of changing a single amino acid in hemoglobin (Section 26-8). The determination of the primary structure of a protein, called amino add or polypeptide sequencing, can help us to understand the protein s mechanism of action. 

A protein is a polypeptide of 50 or more amino acids that has biological activity. The primary structure of a protein is the particular sequence of amino acids held together by peptide bonds. For example, a hormone that stimulates the thyroid to release thyroxin is a tripeptide with the amino acid sequence Glu-His-Pro. Although five other amino acid sequences of the same three amino acids are possible, such as His-Pro-Glu or Pro-His-Glu, they do not produce hormonal activity. Thus the biological function of peptides and proteins depends on the specific sequence of the amino acids. 

Increasing evidence suggests that evolution has used (and is using) the E2 fold for new purposes. In one apparent example of functional expansion, E2 core domains have been observed to be embedded within much larger polypeptide chains [140, 141]. The functional properties of these massive E2s remain poorly characterized, and it is likely that more of them will be discovered. But the clearest case of functional diversification is provided by the UEV proteins. UEVs are related to E2s in their primary, secondary, and tertiary structures, but they lack an active-site cysteine residue and therefore cannot function as canonical E2s [142]. Nonetheless they play several different roles in ubiquitin-dependent pathways. 

Proteins come in many different sizes and shapes. For example, cytochrome c, a protein that transfers electrons, has only one polypeptide chain of 104 amino acids. Yet myosin, the protein that makes muscles contract, has two polypeptide chains with some 2,000 amino acids each, connected by four smaller chains. It is called a multimeric protein. No matter their size, all proteins have a primary, secondary, and tertiary structure. Some also have quaternary structure. 

In contrast with synthetic polymers, proteins are characterized by very high levels of structural order. Unlike synthetic polymers, proteins are characterized by absolutely uniform chain lengths and well-defined monomer sequences (primary structure) [3]. These features are two of the requirements that enable folding of linear polypeptide chains into structurally well-defined and functional proteins. Proteins play an important role in numerous processes in biology, e.g. as carriers for small molecules and ions (examples are presented in Chapter 2.2), as catalysts, or as muscle fibers, and their exquisite properties are closely related to their well-defined three-dimensional structure [3]. 

For the sake of convenience, the different aspects of protein structure have been divided into four categories primary, secondary, tertiary, and quaternary structures. When we speak of the primary structure of a protein, we are concerned with the amino acid sequence of its component polypeptide chains. A protein may have a single polypeptide chain with one N and one C terminus, or it may have two or more polypeptide chains, often termed subunits, with multiple N and C termini. Secondary structure problems address themselves to whether the polypeptide chains of a protein exhibit any sort of periodicity of structure in three dimensions that is, is the polypeptide chain simply an extended ribbon, or is it present in the form of a spring or a folded structure Secondary structure has also been referred to as conformation. Tertiary structure is concerned with the overall three-dimensional appearance of the protein for example, is the shape of the protein molecule best approximated by a sphere or by a disk Last, quaternary structure refers to the number, size, and shape of component polypeptide chains in a protein. 

In describing protein structure it is usual to consider four levels of organization, termed primary, secondary, tertiary and quaternary structure. Primary structure refers to. the sequence of amino acids that makes up the chain of a particular protein (or synthetic polypeptide). Secondary structure is the ordered conformation that the chain (or usually parts of chains) can twist itself into. An example, a section of an a-helical chain is shown in Figure 9.9. More on this shortly. 

We have already discussed primary structure in terms of the general character of amino acids and some specific examples of amino acid sequences in certain proteins will be discussed later. Our attention now is focused on secondary structure, or conformation as we called it when we discussed synthetic polymers. There are a number of factors that afreet the conformation of a polypeptide chain and a lot can be learned initially by just focusing on two of these steric restrictions on bond rotations and the strong driving force for amide groups to hydrogen bond to one another. 

Proteins are basically formed from one or more chains of polypeptides (with a particular primary structure). The chains of amino acids in proteins, being very long, can coil and fold. This spatial arrangement of amino acids is described by the secondary and tertiary structures of proteins. The arrangement of the amino acids that are near one another in the linear sequence is described by the secondary structure. For example, the amino acids may generate a helical structure (a-helix) such that the amino acids chain forms a tridimensional rod, and the amino acids that are four units apart can have hydrogen bonds between their N-H and C=0 groups. An example of a stereo view of an a-helix... 

You do not have to change the primary structure of an enzyme to inactivate it. You can denature a protein. To denature a protein means to cause it to lose its tertiary and quaternary structures so that the polypeptide becomes a random tangle. Mild changes, such as shifts in solvent, temperature, pH, or salinity, may be enough to denature the enzyme. For example, the enzymatic ability to decompose hydrogen peroxide is lost by plant and animal cells when they are heated. 

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 7Te necessary additional infor mation is obtained from overlap peptides (Figure 3.22). A second enzyme is used to split the polypeptide chain at different linkages. For example, chy-motrypsin cleaves preferentially on the carboxyl side of aromatic and some other bulky nonpolar residues (p, 247). Because these chymotryptic peptides overlap two or more tryptic peptides, they can be used to establish the order of the peptides. The entire amino acid sequence of the polypeptide chain is then known. 

Proteins are characterized by their primary, secondary, tertiary, and quaternary structures. The primary structure is the sequence of the amino acids in the polypeptide chain that makes up the protein. Secondary structure refers to the hrst folding of the amino acid chain and reflects, for example, disulfide bonds. Tertiary structure (a monomer) is the final folded configuration of the protein that is controlled by the primary and secondary structures and is thermodynamically driven by the relative hydrophobicity of the component amino acids in the structure. Quaternary structure refers to the functional association of several polypeptides (monomers). For example, the final structure of hemoglobin consists of four associated monomers. Any change in the primary structure of a protein often results in changes to aU the higher level structure. Protein structures must be characterized and controlled during the production process. 

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