PROTEIN AND PEPTIDES

Peptides & Proteins

1. The Peptide Bond

If the amine and carboxylic acid functional groups in amino acids join together to form amide bonds, a chain of amino acid units, called a peptide, is formed. A simple tetrapeptide structure is shown in the following diagram. By convention, the amino acid component retaining a free amine group is drawn at the left end (the N-terminus) of the peptide chain, and the amino acid retaining a free carboxylic acid is drawn on the right (the C-terminus). As expected, the free amine and carboxylic acid functions on a peptide chain form a zwitterionic structure at their isoelectric pH.
By clicking the "Grow Peptide" button, an animation showing the assembly of this peptide will be displayed. The "Show Structure" button displays some bond angles and lengths that are characteristic of these compounds.

The conformational flexibility of peptide chains is limited chiefly to rotations about the bonds leading to the alpha-carbon atoms. This restriction is due to the rigid nature of the amide (peptide) bond. As shown in the following diagram, nitrogen electron pair delocalization into the carbonyl group results in significant double bond character between the carbonyl carbon and the nitrogen. This keeps the peptide links relatively planar and resistant to conformational change. The color shaded rectangles in the lower structure define these regions, and identify the relatively facile rotations that may take place where the corners meet (i.e. at the alpha-carbon). This aspect of peptide structure is an important factor influencing the conformations adopted by proteins and large peptides.

2. The Primary Structure of Peptides

Because the N-terminus of a peptide chain is distict from the C-terminus, a small peptide composed of different aminoacids may have a several constitutional isomers. For example, a dipeptide made from two different amino acids may have two different structures. Thus, aspartic acid (Asp) and phenylalanine (Phe) may be combined to make Asp-Phe or Phe-Asp, remember that the amino acid on the left is the N-terminus. The methyl ester of the first dipeptide (structure on the right) is the artificial sweetner aspartame, which is nearly 200 times sweeter than sucrose. Neither of the component amino acids is sweet (Phe is actually bitter), and derivatives of the other dipeptide (Phe-Asp) are not sweet.
A tripeptide composed of three different amino acids can be made in 6 different constitutions, and the tetrapeptide shown above (composed of four different amino acids) would have 24 constitutional isomers. When all twenty of the natural amino acids are possible components of a peptide, the possible combinations are enormous. Simple statistical probability indicates that the decapeptides made up from all possible combinations of these amino acids would total 20¹⁰!
Natural peptides of varying complexity are abundant. The simple and widely distributed tripeptide glutathione (first entry in the following table), is interesting because the side-chain carboxyl function of the N-terminal glutamic acid is used for the peptide bond. An N-terminal glutamic acid may also close to a lactam ring, as in the case of TRH (second entry). The abbreviation for this transformed unit is pGlu (or pE), where p stands for "pyro" (such ring closures often occur on heating). The larger peptides in the table also demonstrate the importance of amino acid abbreviations, since a full structural formula for a nonapeptide (or larger) would prove to be complex and unwieldy. The formulas using single letter abbreviations are colored red.
The ten peptides listed in this table make use of all twenty common amino acids. Note that the C-terminal unit has the form of an amide in some cases (e.g. TRH, angiotensin & oxytocin). When two or more cysteines are present in a peptide chain, they are often joined by disulfide bonds (e.g. oxytocin & endothelin); and in the case of insulin, two separate peptide chains (A & B) are held together by such links.
The different amino acids that make up a peptide or protein, and the order in which they are joined together by peptide bonds is referred to as the primary structure. From the examples shown above, it should be evident that it is not a trivial task to determine the primary structure of such compounds, even modestly sized ones.
Complete hydrolysis of a protein or peptide, followed by amino acid analysis establishes its gross composition, but does not provide any bonding sequence information.
Partial hydrolysis will produce a mixture of shorter peptides and some amino acids. If the primary structures of these fragments are known, it is sometimes possible to deduce part or all of the original structure by taking advantage of overlapping pieces. For example, if a heptapeptide was composed of three glycines, two alanines, a leucine and a valine, many possible primary structures could be written. On the other hand, if partial hydrolysis gave two known tripeptide and two known dipeptide fragments, as shown on the right, simple analysis of the overlapping units identifies the original primary structure. Of course, this kind of structure determination is very inefficient and unreliable. First, we need to know the structures of all the overlapping fragments. Second, larger peptides would give complex mixtures which would have to be separated and painstakingly examined to find suitable pieces for overlapping. It should be noted, however, that modern mass spectrometry uses this overlap technique effectively. The difference is that bond cleavage is not achieved by hydrolysis, and computers assume the time consuming task of comparing a multitude of fragments.

Structure-Property Relationships

The compounds we call proteins exhibit a broad range of physical and biological properties. Two general categories of simple proteins are commonly recognized.

Fibrous Proteins		As the name implies, these substances have fibre-like structures, and serve as the chief structural material in various tissues. Corresponding to this structural function, they are relatively insoluble in water and unaffected by moderate changes in temperature and pH. Subgroups within this category include:       Collagens & Elastins, the proteins of connective tissues. tendons and ligaments.       Keratins, proteins that are major components of skin, hair, feathers and horn.       Fibrin, a protein formed when blood clots.
Globular Proteins		Members of this class serve regulatory, maintenance and catalytic roles in living organisms. They include hormones, antibodies and enzymes. and either dissolve or form colloidal suspensions in water. Such proteins are generally more sensitive to temperature and pH change than their fibrous counterparts.

1. The Secondary and Tertiary Structure of Large Peptides and Proteins

The various properties of peptides and proteins depend not only on their component amino acids and their bonding sequence in peptide chains, but also on the way in which the peptide chains are stretched, coiled and folded in space. Because of their size, the orientational options open to these macromolecules might seem nearly infinite. Fortunately, several factors act to narrow the structural options, and it is possible to identify some common structural themes or secondary structures that appear repeatedly in different molecules. These conformational segments are sometimes described by the dihedral angles , defined in the diagram on the right below. Most proteins and large peptides do not adopt completely uniform conformations, and full descriptions of their preferred three dimensional arrangements are defined as tertiary structures.

A. Helical Coiling

The relatively simple undecapeptide shown in the following diagram can adopt a zig-zag linear conformation, as drawn. A ball & stick model of this peptide will be displayed by clicking the appropriate button. However, this molecule prefers to assume a coiled helical conformation, displayed by clicking any of the three buttons on the right. The middle button shows a stick model of this helix, with the backbone chain drawn as a heavy black line and the hydrogen bonds as dashed maroon lines. The other buttons display a ball & stick model and a ribbon that defines this -helix. Seven hydrogen bonds, that together provide roughly 30 kcal/mol stability, help to maintain this conformation.
Examine the drawing activated by the middle button. The N-terminal residue (Ala) is on the left, and the C-terminal Gly on the right. The alpha-helix is right-handed, which means that it rotates clockwise as it spirals away from a viewer at either end. Other structural features that define an alpha-helix are: the relative locations of the donor and acceptor atoms of the hydrogen bond, the number of amino acid units per helical turn and the distance the turn occupies along the helical axis. The first hydrogen bond (from the N-terminal end) is from the carbonyl group of the alanine to the N-H group of the phenylalanine. Three amino acids, Thr, Gly & Ala, fall entirely within this turn. Parts of the N-terminal alanine acceptor and the phenylalanine donor also fall within this helical turn, and careful analysis of the structure indicates there are 3.6 amino acid units per turn. The distance covered by the turn is 5.4 Å. Using the dihedral angle terminology noted above, a perfect -helix has  = -58º and  = -47º. In natural proteins the values associated with -helical conformations range from -57 to -70º for , and from -35 to -48º for . To examine a Chime model of this alpha-helix, click on the green circle.
Helical conformations of peptide chains may also be described by a two number term, n_m, where n is the number of amino acid units per turn and m is the number of atoms in the smallest ring defined by the hydrogen bond. Using this terminology, the alpha-helix is a 3.6₁₃ helix. Other common helical conformations are 3₁₀ and 4.4₁₆. The alpha helix is the most stable of these, accounting for a third of the secondary structure found in most globular (non-fibrous) proteins.

B. -Pleated Sheets

The linear zig-zag conformation of a peptide chain may be stabilized by hydrogen bonding to adjacent parallel chains of the same kind. Bulky side-chain substituents destabilize this arrangement due to steric crowding, so this beta-sheet conformation is usually limited to peptides having a large amount of glycine and alanine. Steric interactions also cause a slight bending or contraction of the peptide chains, and this results in a puckered distortion (the pleated sheet). As shown in the following diagram, the adjacent chains may be oriented in opposite N to C directions, termed antiparallel. Using the dihedral angle terminology, an antiparallel -sheet has  = -139º and a  = 135º. Alternatively, the adjacent peptide chains may be oriented in the same direction, termed parallel. By convention, beta-sheets are designated by broad arrows or cartoons, pointing in the direction of the C-terminus. In this diagram, these cartoons (colored violet) are displayed by clicking on the appropriate button. A Chime model of a this three-antiparallel-chain structure may be examined by clicking on the green circle.

The main protein component of silk, fibroin, adopts a beta-pleated sheet secondary structure, a small segment of which may be examined in the Chime model. Silk fibroin is 40-45% Gly, 27-33% Ala, and roughly equal amounts (12-15%) of Ser and Thr. Filaments of silk are particularly thin and uniform in structure.
When beta-sheets are observed as secondary structural components of globular proteins, they are twisted by about 5 to 25º per residue; consequently, the planes of the sheets are not parallel. The twist is always of the same handedness, and is usually greater for antiparallel sheets. Examples will be found in the following structures.

C. Other Structures

Although most proteins and large peptides may have alpha-helix and beta-sheet segments, their tertiary structures may consist of less highly organized turns, strands and coils. Turns reverse the direction of the peptide chain, and are considered to be a third common secondary structure motif. Approximately a third of all the residues in globular proteins are found in turns. Turns occur chiefly on the protein surface, often incorporate polar and charged residues, and have been classified in three sub-groups.
As noted earlier, several factors perturb the organization of peptide chains. One that has not yet been cited is the structural influence of proline. Unlike the other common amino acids, rotation about the  C-N bond in proline is not possible due to the structural constraint of the five-membered ring. Consequently, the presence of a proline in a peptide chain introduces a bend or kink that disrupts helices or sheets. Also, prolines that are part of a peptide chain have no N-H hydrogen bonding donors to contribute to conformer stabilization.
With the exception of silk fibroin and certain synthetically engineered peptides, significant portions of most proteins adopt conformations that resist simple description or categorization. For example, the following diagram shows the tertiary structure of a polypeptide found in cobra venom. A large section of antiparallel beta-sheets is colored violet, and a short alpha-helix is green. The remaining peptide chain seems disorganized, but certain recurring features such as seven 180º turns (beta-turns) and five disulfide bonds can be identified. A Chime model of this compound may be examined by clicking on the diagram.

Additional Examples

A full description and discussion of protein structure is beyond the scope of this text, but a few additional examples will be instructive. In addition to the tertiary structures that will be displayed, attention must also be given to the way in which peptide structures may aggregate to form dimeric, trimeric and tetrameric clusters. These assemblies, known as quaternary structures, have characteristic properties different from their monomeric components. The examples of mellitin, collagen and hemoglobin, shown below demonstrate this feature.
Some proteins incorporate nonpeptide molecules in their overall structure, either bonded covalently or positioned by other forces. These are called conjugated proteins, and the non-peptide components are referred to as prosthetic groups. Examples of conjugated proteins include:

Glycoproteins, incorporating polysaccharide prosthetic groups (e.g. collagen and mucus).
Lipoproteins, incorporating lipid prosthetic groups (e.g. HDL and LDL).
Chromoproteins, incorporating colored prosthetic groups (e.g. hemoglobin).

The seven illustrations shown below identify a set of peptides and proteins that may be examined as Chime models by clicking on a selected picture.

Endothelin & Angiogenin are small peptides that have important and selective physiological properties.
Lysozyme a typical globular protein, incorporating many identifiable secondary structures.
Mellitin, from honey bee venom, has a well-defined quaternary structure, half of which is shown here.
Collagen is a widely distributed fibrous protein with a large and complex quaternary structure. Only a small model segment is shown here.
Thioredoxin is a relatively small regulatory protein serving an important redox function.
Hemoglobin, the most complex of these examples, is a large conjugated protein that transports oxygen. A 170 pound human has about a kilogram of hemoglobin distributed among some five billion red blood cells. A liter of arterial blood at body temperature can transport over 200 mL of oxygen, whereas the same fluid stripped of its hemoglobin will carry only 2 to 3 mL.

Denaturation

The natural or native structures of proteins may be altered, and their biological activity changed or destroyed by treatment that does not disrupt the primary structure. This denaturation is often done deliberately in the course of separating and purifying proteins. For example, many soluble globular proteins precipitate if the pH of the solution is set at the pI of the protein. Also, addition of trichloroacetic acid or the bis-amide urea (NH₂CONH₂) is commonly used to effect protein precipitation. Following denaturation, some proteins will return to their native structures under proper conditions; but extreme conditions, such as strong heating, usually cause irreversible change.
Some treatments known to denature proteins are listed in the following table.

Denaturing Action	Mechanism of Operation
Heat	hydrogen bonds are broken by increased translational and vibrational energy. (coagulation of egg white albumin on frying.)
Ultraviolet Radiation	Similar to heat  (sunburn)
Strong Acids or Bases	salt formation; disruption of hydrogen bonds. (skin blisters and burns, protein precipitation.)
Urea Solution	competition for hydrogen bonds. (precipitation of soluble proteins.)
Some Organic Solvents (e.g. ethanol & acetone)	change in dielectric constant and hydration of ionic groups. (disinfectant action and precipitation of protein.)
Agitation	shearing of hydrogen bonds. (beating egg white albumin into a meringue.)