Peptides & Proteins

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.
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.
3. N-Terminal Group Analysis
Over the years that chemists have been studying these important natural products, many techniques have been used to investigate their primary structure or amino acid sequence. Indeed, commercial instruments that automatically sequence peptides and proteins are now available. A few of the most important and commonly used techniques will be described here.
Identification of the N-terminal and C-terminal aminoacid units of a peptide chain provides helpful information. N-terminal analysis is accomplished by the Edman Degradation, which is outlined in the following diagram. A free amine function, usually in equilibrium with zwitterion species, is necessary for the initial bonding to the phenyl isothiocyanate reagent. The products of the Edman degradation are a thiohydantoin heterocycle incorporating the N-terminal amino acid together with a shortened peptide chain. Amine functions on a side-chain, as in lysine, may react with the isothiocyanate reagent, but do not give thiohydantoin products.
immediately cyclizes to a hydantoin ring, and this can be cleaved from the peptide chain in several ways, not described here. Depending on the nature of this final cleavage, the procedure can be modified to give a C-terminal acyl thiocyanate peptide product which automatically rearranges to a thiohydantoin incorporating the penultimate C-terminal unit. Thus, repetitive analyses may be conducted in much the same way they are with the Edman procedure.

Enzymatic Analysis

Enzymatic C-terminal amino acid cleavage by one of several carboxypeptidase enzymes is a fast and convenient method of analysis. Because the shortened peptide product is also subject to enzymatic cleavage, care must be taken to control the conditions of reaction so that the products of successive cleavages are properly monitored. The following example illustrates this feature. A peptide having a C-terminal sequence: ~Gly-Ser-Leu is subjected to carboxypeptidase cleavage, and the free aminoacids cleaved in this reaction are analyzed at increasing time intervals. By clicking on the diagram, the results of this experiment will be displayed. The leucine is cleaved first, the serine second, and the glycine third, as demonstrated by the sequential analysis. Of course, fourth and fifth units will also be released as time passes, but these products are not shown.

Since end group analysis of large peptides and proteins is of limited value, methods of selectively cleaving such macromolecules into smaller peptide fragments are commonly employed as a major step in structure elucidation. Three selective cleavage methods are outlined in the table on the left. These procedures all cleave peptide chains at designated locations, and at the carboxyl side of the targeted amino acid. A plausible mechanism for the cyanogen bromide cleavage is outlined below. The C-terminal side of the methionine is obtained as a smaller peptide, which can be examined by any of the preceding techniques. The N-terminal side is characterized by a homoserine lactone at its C-terminus.

Mechanisms for the enzymatic reactions are not as easily formulated. Other enzymatic cleavages have been developed, but the two listed here will serve to illustrate their application.

An Example of Primary Structure Analysis

To see how these procedures can be combined to elucidate the primary structure of a peptide, consider the melanocyte stimulating hormone isolated from pigs. This octadecapeptide (18 amino acid units) has the composition: Arg,Asp2,Glu2,Gly2,His,Lys2,Met,Phe,Pro3,Ser,Tyr2, and is abbreviated P18. The following diagram, which begins with the results of terminal unit analysis, illustrates the logical steps that could be used to solve the structural problem. By clicking the "Next Stage" button the results and conclusions from each step will be displayed. Comments about each stage are presented under the diagram.
a) Cyanogen bromide cleavage gives two peptide fragments, the longer of which has all the units on the C-terminal side of methionine.
b) N-terminal analysis of the undecapeptide fragment, P11, locates the three amino acids to the right of methionine.
c) Trypsin cleavage of P11 shows the location of the single arginine, which is found as the C-terminal unit of the tetrapeptide fragment. One of the two lysines was known to be next to the C-terminus. The other must be part of the smaller peptide from the cyanogen bromide reaction.
d) With only four amino acids remaining to be located, the position of the second tyrosine may be pursued by chymotrypsin cleavage of P18 itself. Four fragments are obtained, and the final structure might have been solved by these alone. However, selective terminal group analysis of the two pentapeptides serves to locate the tyrosine and a second proline next to the left most glycine, as well as identifying the units on each side of the methionine. The one remaining amino acid, a proline, is then placed at the last vacant site (yellow box).