4.5. Properties of Peptides
Many of the properties of peptides are similar to those of amino acids. However, the presence of amide functional group(s) and the ability to regularly form three-dimensional shapes make peptides more complex.
4.5.1. Acid/Base Properties of Amides in Peptides
The amide functional group consists of a carbonyl attached to a nitrogen with a lone pair. The lone pair is conjugated to the carbonyl. The nitrogen is slightly more electronegative than the carbon(s) and/or hydrogen(s) it is attached to, making those bonds polar. This combination suggests that amides may be reactive functional groups. However, the opposite is typically true.
Amides are far more robust (less reactive) than may be expected (Figure 4.41). The oxygen of the carbonyl has lone pairs and is, in principle, basic/nucleophilic. However, as with related functional groups (carboxylic acids, esters, etc.) the inductive effect from the nearby electronegative atom (the nitrogen) weakens this; the oxygen of the carbonyl is a weak base/nucleophile. The carbon of the carbonyl is attached to electronegative atoms and is, in principle, electrophilic. However, as with related functional groups (carboxylic acids, esters, etc.) resonance stabilization from the adjacent atom with a lone pair (the nitrogen) weakens this; the carbon of the carbonyl is a very weak electrophile. The nitrogen has a lone pair and is, in principle, basic/nucleophilic. However, as with related functional groups (carboxylic acids, esters, etc.) the lone pair is occupied participating in resonance/conjugation with the adjacent carbonyl and weakens this; the nitrogen of the amide is a very weak base/nucleophile. Finally, if the nitrogen is attached to a hydrogen the bond is polar and it is, in principle, acidic. However, as with related functional groups (amines, etc.) placing an anionic charge on a nitrogen is disfavoured and weakens this; any hydrogen of the amide is a weak acid. Unfortunately, the reasons why anionic charges on nitrogen atoms are disfavoured are complex and outside the scope of this text.
Figure 4.41 – Rationales for Very Low Acid/Base//Electrophile/Nucleophile Reactivity of an Amide Functional Group.
The overall effect is that amides are very stable functional groups and do not generally act as acids/bases/nucleophiles/electrophiles outside of specific conditions.
4.5.1.1. How to Determine a Peptide’s Isoelectric Point (pI)
Like individual amino acids, peptide polymers have isoelectric points. It is possible to use pKa values to determine the isoelectric point of a compound. For peptides there are two methods depending on the nature of the side chains. In both cases this is fundamentally just finding the average between the pKa values that protonate and deprotonate the neutral compound. With peptides the process is simplified because the amides (and the functional groups used to make them) are irrelevant and do not participate in acid-base reactions.
If all of the side chains ARE NOT acidic/basic:
1. Calculate the average of the pKa values for the ammonium of the amino acid at the N-terminus and the carboxylic acid of the amino acid at the C-terminus. This is the pI value. The other ammonium/carboxylic acid pKa values are irrelevant.
As before, this method is simple but obscures what is actually being calculated. The pI value is the average of the two pKa values (viz. pH values) that represent formation of the cationic and anionic forms.
If at least one of the side chains ARE acidic/basic:
1. Draw the molecule as it would exist at a very low pH, with all functional groups in their (conjugate) acid forms.
2. Remove hydrogens from groups one-by-one in ascending order using pKa values. This is the order that they would be removed in solution (most acidic first, least acidic last). It may be helpful to keep track of the net charge on the molecules. Remember to only consider the side chains, the ammonium of the amino acid at the N-terminus, and the carboxylic acid of the amino acid at the C-terminus. The other ammonium/carboxylic acid pKa values are irrelevant.
3. Calculate the average of the pKa values for the shift between the cationic and neutral form and the shift from the neutral to the anionic form. This is the pI value.
Because the calculated pI value will depend on the specific pKa values used different tables may give slightly different results. Unless otherwise indicated, for all pI calculations use the pKa values specified in this text (Figure 4.3).
The formal steps will always give the correct value. However, redrawing all of the structures can be tedious, especially with longer peptide chains. A shortcut is commonly used: once the net charge of the maximally protonated structure is known (Step 4) the pKa values are listed in ascending order with the corresponding net charges increasing by 1 with each deprotonation. The pI value is calculated as usual but without drawing all of the protonation states and only ranking them until the desired pKa,cation and pKa,anion are found.
4.5.1.2. Long-Chain Peptides and Isoelectric Point (pI)
Peptide polymers can be arbitrarily long, with hundreds or thousands of amino acids connected in sequence. While it is possible to calculate isoelectric point values for these chains doing so manually is not feasible. However, the values are often useful and are instead calculated using computers.
The pI values in these cases are heavily impacted by the pKa values of the amino acid side chains. This means that certain peptide chains (with specific side chains to perform specific functions and/or to help the peptide survive certain environmental conditions) may have very characteristic pI values. For example, the pI value for the peptide chain of lysozyme, an anti-microbial enzyme typically found in saliva, is high (ca. 11). This peptide chain has many basic side chains to help it perform its function. Conversely, the pI value for the peptide chain of pepsin, a digestive enzyme typically found in the stomach, is low (ca. 1). This peptide has many acidic side chains to help it survive the acidic environment it is in.
Most commonly, knowing a peptide chain’s distinct pI value is used to help identify an unknown peptide and/or to separate similar peptides (electrophoresis). The specific details of how these techniques work are outside the scope of this text and are instead typically explored in introductory biochemistry courses.
4.5.2. Brief Overview of Peptide Folding
Certain patterns in the ways that long chain peptides fold and create three-dimensional structures are very consistent and observed in the overwhelming majority of cases. These shapes result from sets of amino acids along the chain interacting with each other in regular (repeating) ways. Often this is the result of non-covalent interactions and/or rigidity/flexibility of the chain. While this is heavily influenced by the specific side chains of the amino acids, the exact amino acids required can be highly variable; these shapes can result from MANY different arrangements of amino acids provided they have the required interactions. This is one of the factors that allows biological systems their high degree of flexibility.
Because the exact sequence of amino acids making them up is often irrelevant, these structures are most commonly represented using ribbon-style images. Ribbon-style representations are fundamentally just a simplified view of the “backbone” of the peptide chain (the atoms that make up the main polymer chain; Figure 4.42). Backbone images are not typically used for representing large peptide chains (the ribbon style shows this and additional information). However, to help visualize the exact nature of each structure they will be presented along with a variety of different styles.
Figure 4.42 – Example of a Peptide Chain “Backbone”.
One of the common folding patterns is an alpha- (α-) helix (Figure 4.43). An α-helix occurs when the carbonyls of one amino acid in the chain are able to hydrogen bond well to the N-H’s of the amino acids three positions later; these sections of the peptide chain have good hydrogen bonding between amides from every fourth amino acid of the chain (i.e. the 1st amino acid with the 4th, the 2nd amino acid with the 5th, etc.). The result is that the chain coils back onto itself and forms a spiral shape.
Figure 4.43 – An Alpha- (α-) Helix in Ball-and-Stick, Ribbon, “Backbone Only”, and Overlayed Models with Highlighted Hydrogen Bonding.
All of the amino acids in the peptide are L-amino acids and all of their side chains will “point” in the same direction. To avoid having all of the side chains projecting into the centre of the helix (disrupting the shape) it must form a “right-handed” helix (Figure 4.44): when viewed from above the spiral will proceed clockwise away from the viewer. This is true regardless of which end is considered the “top”. As a result of this all of the side chains point outwards from the spiral.
Figure 4.44 – Side and Top Views of an Alpha- (α-) Helix Showing Right-Handed Nature of the Spiral.
One of the common folding patterns is a beta- (β-) sheet (Figure 4.45). A β-sheet occurs when the side chains of the amino acids in the chain do not allow flexibility of the backbone; these sections of the peptide chain are very rigid because of the side chains of the amino acids in them. The result is that the chain bends very little and forms a linear shape.
Figure 4.45 – A Beta- (β-) Sheet in Ball-and-Stick, Ribbon, “Backbone Only”, and Overlayed Models.
All of the amino acids in the peptide are L-amino acids and all of their side chains will “point” in the same direction. However, because the amino acids “flip” as part of the strand they alternate between pointing directly above and below the sheet (Figure 4.46). In ribbon images β-sheets are typically represented as an arrow pointing in the sequence direction (from the N-terminus to the C-terminus).
Figure 4.46 – Side Chains of a Beta- (β-) Sheet Alternate Projecting Above and Below the Sheet.
Individual β-sheets are uncommon. Typically, sections of the chain that form β-sheets interact with one or more other β-sheets to form larger sheets. Confusingly, these larger sheets (made up of interacting β-sheets) are also referred to as β-sheets; both the individual sections and the larger aggregates are simply called “β-sheets”.
There are two ways that an individual β-sheet may interact with another to form a larger sheet (Figure 4.47). In both cases the amides of one section hydrogen bond to the amides of the other. If the two β-sheets proceed in the same direction (N-terminus to C-terminus) then the combined sheet is called a parallel β-sheet. If the two β-sheets proceed in opposite directions then the combined sheet is called an antiparallel β-sheet.
Figure 4.47 – Parallel and Antiparallel Beta- (β-) Sheets.
Large β-sheets made from multiple interacting individual β-sheets are common. Although individual β-sheets are generally rigid and flat/planar, in some instances the large sheets can curve over longer distances or combine to form even more complex shapes.
One of the common “folding patterns” is a loop (Figure 4.48). A loop occurs when the amino acids in the chain do not interact with each other or other parts of the chain to a significant extent; these sections of the peptide chain have few non-covalent interactions between the amino acid monomers and tend to be highly flexible. The result is that the chain remains loose and does not aggregate to itself or other parts of the peptide.
Figure 4.48 – A Loop in Ball-and-Stick, Ribbon, “Backbone Only”, and Overlayed Models.
Loop sections often have a large number of glycine and proline in them. Glycine does not have a side chain (its R group is hydrogen). This allows glycine to be significantly more flexible than other common amino acids with larger (i.e. sterically bulkier) groups. Conversely, because of its cyclic side chain proline is less flexible than other common amino. However, the ability to shift between s-cis and s-trans conformations makes the chain itself far more flexible.
A small structural feature is sometimes highlighted. A turn is a section of the chain, typically within a loop or between two other structures, where it tightly bends back onto itself (Figure 4.49). A turn occurs when an amide of the chain hydrogen bonds to another very close by, forming a “hairpin” type of structure; these sections of the peptide chain very abruptly turn back on themselves. There are MANY different types of turn structures that vary by the number of amino acids involved, the relative geometries of side chains, etc. Frequently, but not always, there is a proline in the turn to either help the chain fold back on itself (rigidity from the cyclic side chain) or to adopt an s-cis conformation and allow the abrupt change in direction.
Figure 4.49 – Examples of Three Types of Turn Structures.
Depending on the purpose of the image and the software used to generate it, turns may or may not be coloured differently than loops in ribbon-style images. In most cases they are not highlighted and are simply coloured and indicated as loops.
4.5.3. Brief Overview of Peptide Structural Complexity
Peptide polymers are able to adopt a large number of intricate three-dimensional structures which themselves often combine to form larger, even more complex structures. The intricacies of polypeptide folding are VERY complicated, and most institutions have multiple advanced courses dedicated to their study. While this level of discussion is necessary to deal with the topic, at an introductory level it is excessive. In place of giving a complete guide to peptide structural complexity this text will aim to convey the basics to help provide context for later discussions of peptide-derived systems (proteins and enzymes, see Chapter 6).
Generally, discussions of peptide structures divide the topic into four parts.
The primary structure is the sequence of amino acids itself (Figure 4.50). This includes which amino acids are present, the order they are connected in, and may include any simple modifications to the amino acid chain itself (e.g. if the C-terminal is an ester instead of a carboxylic acid). The primary structure does not include any information about the three-dimensional shape, only the connectivity.
Figure 4.50 – Example of a Primary Structure for a Peptide.
The secondary structure is the sequence of local substructures (the sequence of common folding patterns; Figure 4.51). This includes α-helixes, β-sheets, loops, and other common structures not discussed in this text in the order they are connected in. The secondary structure does not include any information about the substructures interacting with each other (e.g. larger β-sheets), only the connectivity of the parts.
Figure 4.51 – Example of a Secondary Structure for a Peptide.
The tertiary structure is the final overall shape that the peptide sequence adopts (Figure 4.39). This is the full three-dimensional structure of the chain accounting for all non-covalent interactions (e.g. hydrogen bonds, π-stacking, hydro-phobic/philic areas, etc.) and any covalent modifications (e.g. disulfide bonds).
Figure 4.39 – Ribbon Model of Thioredoxin 1 (TCF52B).
The quaternary structure is the shape(s) of the overall structure when multiple folded peptide chains (tertiary structures) combine to form a larger structure (Figure 4.52). These are generally very complex systems and are not easily interpreted. Not all peptide chains become part(s) of a quaternary structure. Some quaternary structures are made of multiple copies of the same tertiary structures, some combine entirely different folded chains, and some combine both. Rarely, some peptide chains may be used in multiple different quaternary structures with different functions.
Figure 4.52 – Examples of Quaternary Structures for Peptides.
