Amino acids and proteins Dr Una Fairbrother

Amino acids and proteins Proteins are composed of amino acids Proteins are composed of amino acids When a protein is hydrolysed e.g. with hydrochloric acid then about 20 different amino acids can be detected When a protein is hydrolysed e.g. with hydrochloric acid then about 20 different amino acids can be detected When a protein is sequenced it may be found that it is composed of hundred amino acids When a protein is sequenced it may be found that it is composed of hundred amino acids Therefore each of the different amino acids is occurring more than once Therefore each of the different amino acids is occurring more than once

Each protein has unique sequence Think of coloured beads in a necklace Think of coloured beads in a necklace One necklace may differ from another by the order of the beads and the frequency with which they occur One necklace may differ from another by the order of the beads and the frequency with which they occur Different amino acid content and sequence alters the properties of proteins Different amino acid content and sequence alters the properties of proteins

Illustration of the 20 amino acids

Amino acid game animations.htm animations.htm

General Structure of an alpha amino acid A 'free' amino acid (a single amino acid) always has: An amino group -NH2, A carboxyl group -COOH A hydrogen -H A chemical group or side chain -"R". These are all joined to a central carbon atom, the  -carbon

Only L Amino Acids Are Found in Proteins. The L and D Isomers of Amino Acids. R refers to the side chain. The L and D isomers are mirror images of each other.

R groups Differences between amino acids due to differing R group structures The R group or side chain can be aliphatic, hydroxyl or sulphur containing, aromatic, basic or acidic

Features of the R group The aliphatic amino acids have hydrocarbon side chains which are hydrophobic. The aliphatic amino acids have hydrocarbon side chains which are hydrophobic. They do not like to be in contact with water molecules in an aqueous solution. They do not like to be in contact with water molecules in an aqueous solution. These allow hydrophobic bonding in proteins to occur. These allow hydrophobic bonding in proteins to occur. For this reason, they are often located in the core of the protein, surrounded by the rest of the protein, and "shielded" by them from the aqueous surroundings. For this reason, they are often located in the core of the protein, surrounded by the rest of the protein, and "shielded" by them from the aqueous surroundings.

Properties of the aliphatics Glycine (Gly; G) Glycine (Gly; G) simplest and smallest of all, has a single hydrogen as it's side chai simplest and smallest of all, has a single hydrogen as it's side chai Alanine (Ala; A) Alanine (Ala; A) has a methyl group as it's sidechain. has a methyl group as it's sidechain. Valine (Val; V) Valine (Val; V) Longer sidechain with a branch. As the aliphatic side chains get longer they are also more hydrophobic. Longer sidechain with a branch. As the aliphatic side chains get longer they are also more hydrophobic. Leucine (Leu; L) Leucine (Leu; L) has another methyl group attached to the sidechain. has another methyl group attached to the sidechain. Isoleucine (Ile; I) Isoleucine (Ile; I) similar to L and V but orientation of sidechain atoms is different. Isoleucine also has two centers of asymmetry similar to L and V but orientation of sidechain atoms is different. Isoleucine also has two centers of asymmetry Proline (Pro; P) Proline (Pro; P) Different- the sidechain is bonded to the  -carbon and the amino group. Marked effects on the architecture of proteins. Less hydrophobic than the others. Different- the sidechain is bonded to the  -carbon and the amino group. Marked effects on the architecture of proteins. Less hydrophobic than the others.

Proline Proline is a special case Proline is a special case Due to its structure it causes bends in proteins Due to its structure it causes bends in proteins It is a feature which contributes to the compactness of globular proteins It is a feature which contributes to the compactness of globular proteins

Neutral Polar Amino Acids These amino acids are not charged at physiological pH. These amino acids are not charged at physiological pH. Serine and threonine, asparagine and glutamine have polar hydroxyl groups in their side chains Serine and threonine, asparagine and glutamine have polar hydroxyl groups in their side chains These can contribute to hydrogen bonding in proteins These can contribute to hydrogen bonding in proteins For this reason the amino acids are classed as hydrophillic. For this reason the amino acids are classed as hydrophillic.

Sulphur containing amino acids Cysteine (Cys; C) Cysteine (Cys; C) contains a thiol or sulphydryl group (-SH). contains a thiol or sulphydryl group (-SH). This is extremely reactive, and can form hydrogen bonds. This is extremely reactive, and can form hydrogen bonds. But the long aliphatic part of the side chain makes it quite hydrophobic. But the long aliphatic part of the side chain makes it quite hydrophobic. The (-SH) group of cysteine can be oxidised with another cystein -SH to give a -S-S-disulphide bridge The (-SH) group of cysteine can be oxidised with another cystein -SH to give a -S-S-disulphide bridge Methionine (Met; M) Methionine (Met; M) is a very special amino acid in that it is the "start" amino acid in the process of translation (protein synthesis). is a very special amino acid in that it is the "start" amino acid in the process of translation (protein synthesis). sulphur atom in a thioether linkage, and is relatively unreactive. sulphur atom in a thioether linkage, and is relatively unreactive. Has a highly hydrophobic sidechain. Has a highly hydrophobic sidechain.

Aromatic amino acids Two of the aromatic amino acids, tryptophan and tyrosine are useful in quantitative protein determination. Two of the aromatic amino acids, tryptophan and tyrosine are useful in quantitative protein determination. They are detected in the Lowry assay: a biochemical test to measure the amount of protein in a sample. They are detected in the Lowry assay: a biochemical test to measure the amount of protein in a sample. Copper(II) ion in alkaline solution reacts with protein to form complexes, which react with a Folin-phenol reagent. The product becomes reduced and can be detected colourimetrically by absorbance at 650 nm. Copper(II) ion in alkaline solution reacts with protein to form complexes, which react with a Folin-phenol reagent. The product becomes reduced and can be detected colourimetrically by absorbance at 650 nm.

Aromatic amino acids Contain an aromatic ring as part of their sidechains. Contain an aromatic ring as part of their sidechains. Because of the aromatic rings, they are highly hydrophobic Because of the aromatic rings, they are highly hydrophobic Phenylalanine (Phe; F) Phenylalanine (Phe; F) Contains a phenyl ring attached to a methylene group Contains a phenyl ring attached to a methylene group Tyrosine (Tyr; Y) Tyrosine (Tyr; Y) contains a hydroxyl group on the phenyl ring contains a hydroxyl group on the phenyl ring makes it less hydrophobic than F. It is also a reactive group makes it less hydrophobic than F. It is also a reactive group Tryptophan (Trp; W) Tryptophan (Trp; W) has a slightly different ring attached to the methylene group. has a slightly different ring attached to the methylene group. This is an indole ring and it is highly hydrophobic. This is an indole ring and it is highly hydrophobic.

Charge The basic and acidic amino acids can exist in charged forms either -ve (with acidic aas) or +ve (with basic aas). The basic and acidic amino acids can exist in charged forms either -ve (with acidic aas) or +ve (with basic aas). Attraction between oppositely charged amino acids in proteins produces ionic bonds. Attraction between oppositely charged amino acids in proteins produces ionic bonds.

Acidic Amino Acids These amino acids are highly polar, and are nearly always negatively charged at physiological pH. These amino acids are highly polar, and are nearly always negatively charged at physiological pH. Aspartate (Asp; D) Aspartate (Asp; D) is really aspartic acid. is really aspartic acid. It is called aspartate because it is usually negatively charged at physiological pH and so it is named for the carboxylate anion. It is called aspartate because it is usually negatively charged at physiological pH and so it is named for the carboxylate anion. Glutamate (Glu; E) Glutamate (Glu; E) is also called glutamic acid. is also called glutamic acid. The side chain of glutamate also has a carboxylate group which has a negative charge at physiological pH The side chain of glutamate also has a carboxylate group which has a negative charge at physiological pH

Basic Amino Acids Contain side chains which are postively charged at physiological pH. Contain side chains which are postively charged at physiological pH. Lysine (Lys; K) Lysine (Lys; K) one of the longest side chains one of the longest side chains it is very polar because of the terminal amino group and is a hydrophillic amino acid. it is very polar because of the terminal amino group and is a hydrophillic amino acid. Arginine (Arg; R) Arginine (Arg; R) the largest of all sidechains the largest of all sidechains Bacause of the guanidino group on the sidechain it is postively charged at physiological pH. Bacause of the guanidino group on the sidechain it is postively charged at physiological pH. Histidine (His; H) Histidine (His; H) has an imidazole ring which often sits inside the active site of an enzyme and helps bonds to be broken or made. has an imidazole ring which often sits inside the active site of an enzyme and helps bonds to be broken or made. It can do this because it can exist in two states -uncharged, or positively charged. It can do this because it can exist in two states -uncharged, or positively charged.

Ion Pairs When amino acid sidechains of opposite charge are in close proximity, they can form an ion pair (also called a salt bridge). When amino acid sidechains of opposite charge are in close proximity, they can form an ion pair (also called a salt bridge). If they can form a salt bridge, they will usually be buried. If they can form a salt bridge, they will usually be buried. Since charge is affected by pH, so is the formation and the breakage of these ion pairs. Since charge is affected by pH, so is the formation and the breakage of these ion pairs. Salt bridges increase the stability of the tertiary structure Salt bridges increase the stability of the tertiary structure.

Features of the  -amino and  carboxyl group In solution amino acids can exist in different charged forms depending on pH. In solution amino acids can exist in different charged forms depending on pH. At the most acid pH values they are fully protonated with two weakly acidic groups. At the most acid pH values they are fully protonated with two weakly acidic groups.

Dissociation constant (pKa) values If we know the pKa value for a protein titratable group, we can predict the charge present on this group if the protein is in solution at a given pH value, since pH=-log([H 3 O+]) If we know the pKa value for a protein titratable group, we can predict the charge present on this group if the protein is in solution at a given pH value, since pH=-log([H 3 O+]) Glycine contains two ionisable groups: an  -carboxyl group and a protonated  -amino group. Glycine contains two ionisable groups: an  -carboxyl group and a protonated  -amino group. As base is added, these two groups are titrated. As base is added, these two groups are titrated. The pKa of the  -COOH group is 2.4, whereas that of the  -NH3+ group is 9.8. The pKa of the  -COOH group is 2.4, whereas that of the  -NH3+ group is 9.8. The pKa values of these groups in other amino acids are similar. The pKa values of these groups in other amino acids are similar. Some aas, such as aspartic acid, have an ionisable side chain. Some aas, such as aspartic acid, have an ionisable side chain. The pKa values of ionizable side chains in amino acids range from 3.9 (aspartic acid) to 12.5 (arginine). The pKa values of ionizable side chains in amino acids range from 3.9 (aspartic acid) to 12.5 (arginine).

Zwitterion At a neutral pH, both the amino and the carboxyl groups are ionised giving what is termed the zwitterionic form of the molecule (or dipolar ion).

Titration of the  -Carboxyl and  - Amino Groups of an Amino Acid Titration of the  -Carboxyl and  - Amino Groups of an Amino Acid. First the carboxyl group then the protonated amino group loses its proton as the pH increases.

Ionisation State as a Function of pH: the graph The ionisation state of amino acids is altered by a change in pH. The zwitterionic form predominates near physiological pH.

Titration with NaOH The curve produced is really two curves added together. On the left-hand side is the curve from the titration of the carboxyl group. On the right-hand side is the curve from the titration of the amino group. The curve produced is really two curves added together. On the left-hand side is the curve from the titration of the carboxyl group. On the right-hand side is the curve from the titration of the amino group. One equivalent of NaOH is required to titrate each acidic group. One equivalent of NaOH is required to titrate each acidic group. When the first equivalent has been added the amino acid is in its dipolar form. When the first equivalent has been added the amino acid is in its dipolar form.

Question What are the relative amounts of structure I and structure II when 0.5 equivalents have been added? There are equal amounts of each

Hydrogen Bonds When unfolded, all polar/hydrophillic sidechains can interact via H- bonds with water. When unfolded, all polar/hydrophillic sidechains can interact via H- bonds with water. When the protein folds, they must H-bond to each other and exclude much of the water. When the protein folds, they must H-bond to each other and exclude much of the water. All groups capable of forming a hydrogen bond MUST, hence H- bonding in the backbone (C=O to N-H) by way of helices and sheets is an efficient way of ensuring maximum H-bonding. All groups capable of forming a hydrogen bond MUST, hence H- bonding in the backbone (C=O to N-H) by way of helices and sheets is an efficient way of ensuring maximum H-bonding. Sidechains can either accept (as in C=O) or donate (as in N-H, or O- H) an H-bond. Sidechains can either accept (as in C=O) or donate (as in N-H, or O- H) an H-bond. The capacity of proteins to form hydrogen bonds is an important determinant of protein stability. The capacity of proteins to form hydrogen bonds is an important determinant of protein stability. Hydrogen bonds can be: Hydrogen bonds can be: between backbone groups, as in helices and sheets; between backbone groups, as in helices and sheets; between side chains, such as serine or threonine O-H groups and carbonyl carbons of side chains (-C=O); between side chains, such as serine or threonine O-H groups and carbonyl carbons of side chains (-C=O); and between backbone groups and side chain groups. and between backbone groups and side chain groups.

Summary General features General features Aliphatic amino acids Aliphatic amino acids Neutral polar amino acids Neutral polar amino acids Sulphur containing amino acids Sulphur containing amino acids Aromatic amino acids Aromatic amino acids Charge: Charge: Acidic amino acids Acidic amino acids Basic amino acids Basic amino acids Ion pairs Ion pairs pKa values pKa values Titration with NaOH Titration with NaOH Hydrogen bonds Hydrogen bonds Question Question

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