Amino Acids, Peptides and Proteins

CP2 – Semester 2

Isoelectric Points• The isoelectric point of an amino acid is the pH at which the molecule is most neutral

in terms of charge and therefore most likely to be absorbed through plasma membranes.

• The pI of an amino acid can simply be calculated by adding the pKa values of the COOH and NH2 where the side group is not ionisable. However, in many cases the side group contains a functional group which is ionisable.

• Assuming that the pKa of the functional groups is known, a table can be constructed that allows determination of the pI of the molecule. A pH should be chosen that is lower than all pKa values, one that is higher than all pKa values, and then pH values for in-between each pKa. In each box, the ionisation state of the functional group should be detailed, allowing the overall molecular charge to be calculated. An example is given on the next slide.

• The isoelectric point can be calculated from the two pKa values lying either side of the pH at which the overall charge is 0.

Isoelectric Points

pH COOHpKa = 2.2

NH2pKa = 9.0

NH2 SidepKa = 10.5

Overall charge

0 0 + + +25 - + + +110 - 0 + 012 - 0 0 -1

pI = (9.0 + 10.5)/2 = 9.75

Optical Activity of Amino Acids

• The NCC backbone of amino acids allows for them to have optical isomers, all except for glycine, which does not have four different groups attached to the carbon and so cannot form optical isomers.

• In regular chemistry, the S isomer is the one in which, when arranged with the H going away from the observer, groups increase in molecular weight in an anti-clockwise direction. R isomers are those in which when arranged in the same way will have the groups increasing in molecular weight in a clockwise direction.

• Nomenclature for amino acids uses “L” and “D” notation, which roughly equates to “S” and “R”.

• All amino acids capable of forming chiral centres are in the L form except for cysteine, which exists as as the R isomer.

• The carbon at the centre of the NCC backbone is referred to as the α-carbon.• In the natural synthesis of proteins, L amino acids are always used; those containing

D amino acids have been generated post-translation. As the L form of amino acids (the S form) is native to cells, designing proteins with these amino acids results in quicker degradation.

• As such, D amino acids have a greater intracellular stability and are less prone to degradation than their constituent L amino acids.

α and β Amino Acids• Regularly, amino acids will have the NH2 and COOH groups attached to the same C in

the NCC backbone; these amino acids are referred to as α amino acids. • Alpha amino acids are native to cells and so, in exactly the same manner that L

amino acids, are degraded quicker than beta amino acids; beta amino acids are better for drug molecules.

• Beta amino acids are those in which the NH2 and COOH groups are not attached directly to the same carbon, i.e. the backbone is not NCC and is instead NCCC or similar.

• Beta amino acids are very uncommon, but are more stable than alpha amino acids and are not degraded by proteases.

Peptide Synthesis• A peptide is a sequence of amino acids less than 40 AA’s in length, whilst those

longer than 40 AA’s are considered to be proteins.• When reacting a COOH and an amine, the carboxylate must first be activated by

converting it to an acid chloride or an anhydride. These structures have a carbonyl component with a C atom that can be attacked by the nucleophilic N atom of an amine.

• This is because chlorine is a poorer leaving group, or in the case of anhydrides, because of the resonance forms.

• The activation of the carboxylate can be achieved through reacting with thionyl chloride (SOCl₂)

• Synthesising peptides can be complicated because of the potential for molecules of the same amino acid to react with each other and form unwanted by-products.

• The formation of unwanted by-products can be avoided by protecting the amines that aren’t intended for reaction, through the addition of a protecting group.

• The protecting group added to the amino acid must make the amine a poorer nucleophile, so that it has a lower tendency to react with members of it’s own species. The choice of protecting group is important as it must be protecting but still be able to be removed without disturbing new amide bonds formed after synthesis.

Protecting Groups• The three main chemical moieties used to protect amine groups on amino acids are

benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc).

• The result of protecting amines with these groups is that they are unreactive towards acyl chlorides and can be removed under mild conditions that don’t affect peptide bonds.

• Z-group can be removed via catalytic hydrogenation or HBr in acetic acid. As HBr is a dangerous gas, it can be bubbled through acetic acid solution or supplied in a 35% solution of acetic acid.

• Boc can be removed through HCl treatment, or CF₃COOH in acetic acid.• Fmoc can be removed under basic conditions, often with piperidine due to high basicity.

Z Boc Fmoc

Removing Fmoc• Fmoc is a protecting group that can be removed through reaction with a strong nucleophilic

base like piperidine. • Piperidine is a strong nucleophile because the formation of a cationic N atom is stabilized by

the electron donating nature of the alkyl ring in which the N atom sits. This means that the molecule favours the acceptance of a hydrogen and is a powerful dehydrogenating agent.

1. To begin, the piperidine strips the CH₂ of a H atom to form a cationic piperidine moiety and an unstable anionic CH-

component. The unstable CH- component then collapses via the

formation of a pi bond, which causes a double bond to form between the neighbouring carbon and oxygen.

2. This carbon would have 5 bonds and therefore donates a pair to the N atom as this has a lower electronegativity than the oxygen atom, forming carbon dioxide and an anionic amine.

3. This anionic amine is unstable, and so strips a H atom from the charged piperidine moiety to reform piperidine, and form a stable primary amine.

4. The piperidine is then free to act as a strong nucleophile again, and so attacks the free carbon participating in the pi bond. This causes collapse of the pi bond and the formation of an anionic carbon. As the N is forming 4 bonds, it has a positive charge and so the anionic carbon strips a H atom from the N atom to confer neutrality to the whole molecule, and forming the final product of the reaction.

Protecting Side Groups• Amino acids with side groups that contain hydroxyl components (tyrosine, threonine,

serine) must have their OH protected as they act as a site of phosphorylation and consequent modification of the peptide.

• Protecting the amine group of amino acids is also necessary in automated peptide synthesis because of the tendency for the amine to react with R-Cl residues and exposed carboxylates.

• Tyrosine residues are of particular importance intracellularly, as they exist on the IC side of some cell-surface receptors, becoming phosphorylated to start an IC cascade. This can be a target for anti-cancer therapy as interfering with this pathway can stop cells from working; an effect which is seen in greater amounts in cancer cells.

• Proteins that are tagged for degradation have surface lysine residues bonded to ubiquitin to form unbranched polyubiquitin tails. These peptides are then destroyed by proteasomes.

• Monoubiquitination of lysine residues causes transport and localisation within the cell. • Inhibiting the proteasome that is responsible for degrading proteins marked for

disposal has proven to be an interesting anti-cancer target as it kills cancer cells far faster than normal somatic cells. This can be thought to be the result of increased protein assembly in cancer cells due to rapid proliferation, which causes excess protein to build up in the cells and resultant apoptosis.

Automated Peptide Synthesis

• Manual synthesis takes a long time, and so an automated method (the Merrifield method, devised by Robert B. Merrifield) was developed. Synthesis occurs in a C – N direction only.

• This method involves the use of a polystyrene resin with many CH₂Cl residues on the surface.

1. The first amino acid is passed through the column, where the Cl reacts with carboxylate residues of the C-terminal to anchor them to the solid support. This occurs in the presence of OH- which activates the carboxylate.

2. The system is the flushed and purified to remove unreacted amino acid, leaving behind a solid resin support to which one species of amino acid is attached at the C-terminus.

3. The protecting group found on the N-terminus of the amino acids is then removed via a suitable pathway (i.e. CF3COOH for Boc, piperidine for Fmoc) to expose the amine group.

4. The system is flushed and purified to remove the lingering protecting groups.5. The second amino acid in the peptide sequence is then added to the column with a

coupling agent such as DCC (a carbon di-imide), to form the first peptide bonds. The amine group on these amino acids will also be protected to prevent self-association.

6. The system is then flushed and purified to remove unreacted amino acid, then the protecting group is removed, and the system flushed again, leaving behind a dipeptide attached to a solid support, to which further successive peptides can be added following the same process, to a maximum of 70 – 80 amino acids.

Native Chemical Ligation• This is a bio-orthogonal (exclusive/specific) method than can be used to produce

peptides with amino acid chains less than 200 in length, with almost 100% efficiency. The reaction occurs between the N-terminal thiolate of a cysteine amino acid, and the thioester of another, unprotected peptide. This is carried out at pH 7, at 20°C – 37°C in aqueous buffer.

• This method is advantageous as no protecting groups need to be added or removed.

1. The reaction begins when the thiolate S lone pair attacks the unprotected carbonyl. The sulphur is a stronger nucleophile than N in this case.

2. As always occurs when a carbonyl group is attacked by a nucleophile, a tetrahedral intermediate forms with an anionic oxygen, which then collapses to reform the carbonyl, and kicks out the best leaving group. In this case, the S-R component is weakest and so the electrons from the C-S-R bond strip the H atom from the neighbouring S atom (which just attacked the carbonyl) as this S has developed a positive charge.

3. The result of this is that the carbonyl is reformed and a new S-Amine has been attached to the carbonyl molecule. A thiolate molecule is also formed.

4. Due to the proximity of the cysteine’s N-terminus to the carbonyl now, it is able to attack the carbonyl to form a new tetrahedral intermediate which collapses to confer electrons to the S atom, which strips a H from the cationic N atom.

5. This forms the amide bond and reforms the original thiolate component of the cysteine.

Edman Degradation• Edman degradation/sequencing is a method of determining peptide sequences via

systematic cleaving of individual amino acids from a peptide.• This method is only efficient to segments of 30 amino acids or less in size, and so

polypeptides will need to be chopped up before this can be used. However, this remains the most powerful technology to use.

• A component is attached to the N-terminus of the polypeptide, which then cleaves this amino acid and re-attaches to the next residue. In this fashion, amino acids can be removed one at a time, assayed via analytical methods, and thus a sequence can be built.

• The reagent used in Edman sequencing is phenyl-thiocyanate.• Because of the ability only to cleave smaller peptides, other methods have been

used. Chymotrypsin and trypsin are enzymes that are routinely used to cleave larger peptides into smaller fragments. They work at certain sites along a peptide chain.

• Trypsin works at the C-terminus of basic amino acids, like R and K, whilst chymotrypsin works at F, Y, W, L, M, N and Q.

Ribosomal Peptide Synthesis

• Ribosomal peptide synthesis is what occurs in vivo, via ribosomes made of two subunits. The tRNA molecules bear an anticodon segment which allows binding to mRNA for formation of the peptide bond between the two adjacent amino acids.

• The amino acid is activated in biosynthesis through conjugation of adenosine monophosphate at the 3’-hydroxyl, which involves ATP.

• The resulting amino-acyl AMP is an anhydride that then reacts with the 3’-hydroxyl of the terminal tRNA.

• Ribosomal synthesis does not necessitate the use of protecting groups

Non-ribosomal Peptide Synthesis• The synthesis of peptides without ribosomes occurs independently from mRNA, via

peptide synthases. This occurs most commonly in fungi and bacteria, and the individual peptide synthases only synthesise specific peptides.

• The structures of peptides synthesised via peptide synthases are complicated and diverse, due to the ability to incorporate branched, cyclic and D-isomer amino acids, as well as glycosylated amino acids. Ribosomes do not accept D-amino acids.

• Vancomycin (an antibiotic) and cyclosporine (an immunosuppressant) are examples of complicated peptides synthesised via non-ribosomal means.

• Peptide synthases are organised into separate modules that are each responsible for the addition of a specific amino acid. The amino acids are first activated by ligation to AMP, which involves ATP. The aminoacyl-AMP is converted to a thioester by reacting via the thiol group in enzymes.

• Each module will usually contain an adenylation domain (A), a peptidyl carrier domain (PCP) and a condensation/elongation domain (C).

• The thioester linkage is not a cysteine residue, but a pantetheinyl arm (PTA) that is formed of pantothenic acid (vitamin B5) linked to cysteamine via an amide bond.

Protein Structure - Primary

• Peptide synthesis results in the formation of polypeptides; a sequence of amino acid residues which comprise the primary structure of a protein. Cysteine residues are capable of forming bonds within this primary structure through the presence of thiols on their side group which can become oxidised to form disulphide bridges.

• The primary structure of proteins can be seen when the secondary structure is broken, with HPLC being used to separate out different polypeptides.

• B-mercaptoethenol is a reducing agent that can be used to cleave the disulphide bridges between cysteine residues.

• The primary structure is important, because the overall tertiary/quaternary structure of peptides is influenced the by nature of the NCC backbone, which is itself influenced by the nature of side groups.

• There is little freedom of movement because in every AA, the C, O and N are sp² hybridised which allows resonance of the double bond between the C atom and each of the O and N atoms.

• For stabilisation, the p orbital of N lines up with the C=O bond, allowing the formation of a partial C=N bond that prevents rotation and induces rigidity within the structure.

Protein Structure - Primary

• The formation of the partial C=N bond allows the formation of cis and trans isomers. • When unfolded, amino acids may adopt either configuration, however once folded

the trans state is more common as it is preferred energetically. This is because of lack of steric clash between R groups/side chains when they are positioned oppositely across the double bond.

• This trans conformation means that R groups are always seen to be positioned alternating across the backbone to avoid steric clashes.

• The cis conformation positions both R groups on the same side of the double bond. • Despite the presence of chiral centres which are capable of rotation, the coplanar CO

and NH groups allow for extensive hydrogen bonding, that forms the secondary structure of the protein.

Protein Structure - Secondary• The interaction between parallel CO and NH groups means that a secondary structure

is adopted by peptide chains, which is called the formation of β-sheets. • These sheets can be parallel or antiparallel. Parallel sheets are formed of peptide

chains where the amino peptide linkages travel in the same direction. This arrangement is not favoured as it is harder to achieve and forces the chains to bend slightly, forming pleats so that side chain interactions are minimised.

• Antiparallel β-sheets are more common as the amino peptide linkages run in opposite directions and can bond much more easily without steric clashes.

• β-sheets can involve hydrogen bonds between parallel peptide chains (intermolecular) or within the same chain (intramolecular). In order for chains to form intramolecular bonds, the chain must be sufficiently long to fold back on itself.

• When a chain folds back on itself, a β-turn is formed; this is always formed of 4 amino acids. The four amino acids only hydrogen bond between the n and the n + 3 amino acid residues. Proline, because of the incorporation of it’s cyclic side group into the NCC backbone, naturally introduces a bend into the chain. It has no NH₂ component and so is unable to form hydrogen bonds.

Protein Structure - Secondary• β-sheets are one secondary structure option that a polypeptide may assume,

however α-helices are more common.• An alpha helix is a right-handed coiled structure that is stabilised by hydrogen bonds

between CO and NH constituents of the same chain, as well as disulphide bridges. There are 3.6 amino acids per alpha helix turn.

• R groups in an alpha helix orient themselves outwards to preserve the structure (except for proline).

Protein Structure - Tertiary

• The further arrangement of the polypeptide into a 3D structure of folds constitutes the tertiary structure of the protein. The conformation of the protein is affected by non-covalent intermolecular bonds such as hydrophobic interactions and hydrogen bonds, as well as some covalent disulphide bridges.

• The 3D shape of a protein may change according to the nature and pH of the environment. In aqueous environments, the hydrophobic elements will be shielded in the centre of the protein, whilst in non-aqueous environments, hydrophilic side groups will orient inwards to achieve optimal interfacial tension.

• Hydrogen bonds are common contributors to the tertiary structure of proteins, which don’t have to involve CO and NH moieties. Below are some examples.

Protein Structure - Tertiary

• The pH of the environment is an important factor, as some amino acids possess residues that are ionised at physiological pH ranges, which means that there is the potential for rearrangement of structure and also interaction between oppositely charged ionised groups.

• This interaction between charged groups can occur internally to contribute to structure, though charged groups are often seen on the outer surface of proteins where their interaction with the aqueous media is energetically preferable.

• Glutamic and aspartic acid both contain carboxylic acid residues that are deprotonated at pH 7, whilst lysine and arginine both contain N atoms that are protonated at pH 7.

Enzymes• Enzymes are biological catalysts that are capable of increasing reaction rates by 10⁶ -

10¹², with specificity towards substrates. The lock-and-key hypothesis suggests complementarily shaped substrates that fit into the active site of enzymes, though the induced-fit hypothesis provides a better representation, which accounts for a slight conformational change that is induced in the enzyme structure upon formation of the enzyme-substrate complex.

• The interactions between enzyme and substrate are the same non-covalent interactions formed between proteins themselves; Van der Waal’s, hydrogen, hydrophobic interactions etc.

• Enzymes will only accept substrates of specific stereoisomerism; those that bind proteins only bind L-amino acids, whilst those that metabolise sugars only bind D-sugars.

• Enzymes do not however always have the same geometric specificity. Carboxypeptidase-A hydrolyses the C terminal of all polypeptides as long as the penultimate amino acid is not proline, arginine or lysine, and as long as the second one in is also not proline.

• Chymotrypsin catalyses the hydrolysis of esters and amides, and so although being stereospecific, is not geometrically specific.

Enzymes - Lysozyme

• Lysozyme is a protein made of 129 amino acids residues, with a primary structure that features four disulphide bridges. It’s tertiary structure contains alpha helices, disordered loops, antiparallel beta sheets and beta turns. It catalyses the hydrolysis of glycosidic bonds in oligosaccharides, causing bacterial cell wall damage. This leads to compromised integrity of the wall, causing bacteria to burst under their own internal pressure.

• Above is the Richardson/Ribbon structure of lysozyme.

Enzymes - Protein Kinases• Protein kinases are common and there are over 500 types in the body. They

phosphorylate groups on amino acid residues serine, threonine and tyrosine (also histidine less commonly). Some are specific and others are less specific. Phosphorylation of residues is reversible and can undone via hydrolysis of the phosphate groups from amino acids via phosphatase enzymes.

• Protein kinases play a role in cell signalling, metabolism, cellular transport and protein regulation. As there are much more kinases in the body than there are phosphatases, they are a more desirable target for drug delivery as they are more specific and will affect less downstream processes if targeted.

• Successive phosphorylations to the alpha phosphoryl group create beta and gamma groups.

• Phosphorylation can activate or deactivate enzymes and kinases can themselves be activated or deactivated through phosphorylation, autophosphorylation or binding of an allosteric modulator. Allosteric modulators bind to a site on the protein other than the active site, changing the structure; this is a common way to regulate activity.

• Enzymes involved in intracellular cascades (including kinases) are not always that specific, as demonstrated by Akt, which has 30/40 substrates with many process regulating Akt action, which itself then regulates many more processes.

• Targeting of intracellular kinases or enzymes is more preferable than cell surface receptors as it is associated with fewer downstream side effects.

Enzymes - Protein Kinases• Mutation of kinases is common in disease and inhibition of kinases is a drug target.• Inhibitors of kinases can be divided broadly into those which compete with the

substrate for the active site of the enzyme (competitive inhibitors) and those that bind elsewhere on the enzyme to cause a conformational change that deforms the active site and so deactivates the enzyme (allosteric inhibitors).

• However, both allosteric and competitive inhibitors of protein kinases belong to a group of inhibitors called direct inhibitors. This means that these molecules are able to exert their inhibition through directly binding to the enzyme and affecting the formation of enzyme-substrate complexes in this manner.

• The other inhibitors do not bind directly to the enzyme and instead bind to the substrate to prevent the formation of enzyme-substrate complexes via these means.

Enzymes – Ubiquitin Ligases• Ubiquitin is a small (8.5kDa) protein that is found in most cells. Attachment of ubiquitin to a

protein is called ubiquitination, which has many effects including; gene expression, change of enzymatic activity, change of peptide-peptide interaction, change of cellular location and degradation through the proteasome.

• Chemically, ubiquitination is the attachment of the terminal glycine residue of ubiquitin to the lysine residue from a substrate. This results in the formation of an isopeptide bond, which is a peptide bond in a polypeptide, not belonging to the backbone of the protein. This is reversible.

• Ubiquitination can be monoubiquitination or polyubiquitination, each with a different meaning. • Balance of proteins is carried out by the ubiquitin proteasome system (UPS), which seeks and

destroys excess/damaged proteins. UPS failure leads to disease.• When the UPS becomes overactive, useful proteins are destroyed. Restraining of the system

results in growth of excess proteins. UPS disorders have been implicated in cancers, Alzheimer’s, inflammatory diseases like rheumatoid arthritis and also infectious diseases.

• Ubiquitin cannot attach at random to proteins; it must be primed by E1 (activating enzymes) via ATP. This activated ubiquitin is transferred to E2 (conjugating) enzyme, which shuttles it to E3 (ligase) enzyme. E3 acts as a platform for interaction between substrate for degradation and E2/ubiquitin shuttles, which are bought close together by E3.

• When the polyubiquitilated protein interacts with the proteasome, the chain is cleaved and recycled. The protein passes through the chamber of the proteasome where AAs are cleaved.

Enzymes – Ubiquitin Ligases

• Above is the Richardson/Ribbon structure of proteasome.• Ubiquitin can be removed from proteins via deubiquitinating enzymes (DUBs) which

either cleave the peptide or isopeptide bond between ubiquitin and substrate protein.• The proteasome is also a target for drug therapy, with one drug (Bortezomib/Velcade)

inhibiting the action of proteasomes non-specifically. It is licensed for use in the treatment of some cancers. Its application here is probably through the fact that cancerous cells will be producing far more proteins than normal somatic cells, and so inhibition of the proteasome in these rapidly dividing and mutating cells will lead to death quicker than in healthy cells.