Engineering Proteins (EP)

EP1 – Christopher’s Story.

Diabetes;

· The pancreas does not produce Insulin.

· Insulin is the hormone responsible for controlling the uptake of glucose and other sugars by our cells.

· Diabetics must inject insulin into their bodies – or would die.

· Diabetes is controlled by;

§ Carefully monitoring blood glucose levels.

§ Injections of insulin.

§ Strict regulation of diet and regular exercise.

More about Insulin.

· Insulin is a hormone and a protein (Some hormones are proteins, others are not). Proteins also perform many other functions in the body – see fig.4 page 138.

· Proteins are large molecules, natural polymers, with Rmm values up to about 100 000.

· Originally pig or cow insulin used to treat diabetics.

· Today synthetic human insulin available.

Splitting the six-pack.

· The graph below shows how the concentration of insulin in the body of a non-diabetic person changes after a meal.

FIG 6 Page 139

· Notice these 4 important points;

· Maximum concⁿ is low, but this has a profound effect.

· There is always a low-level background concⁿ of insulin.

· Insulin release follows rapidly after eating.

· Insulin concⁿ peaks soon after eating then falls off back to background level.

· The body makes insulin all the time and stores it in special cells in the pancreas. It is not stored as individual molecules, but as hexamers – six molecules clustered together because of interactions between their surfaces.

· As concⁿ of glucose in blood rises insulin hexamers released into bloodstream. As insulin spreads out and becomes more dilute the hexamers

burst apart to form dimers. On further dilution these split into monomers, which can be quickly carried to where they are needed.

0-----------------------------------------------------® 30 Time after

injection/min

Hexamer Dimers Monomers

Concⁿ/

1 x 10^-31 x 10^-6 1 x 10^-8 Mol dm^-3

Injecting Insulin

· Diabetics must wait ½ an hour between injecting insulin and eating.

· The graph below compares insulin levels in the blood of a normal person with those of a diabetic after injecting insulin.

Fig 7 Page 139.

· Diabetics must inject insulin as hexamers, but these are too big to pass easily into the blood stream.

· They must spread out from injection site to become small enough to break up into dimers then monomers (which can pass into blood)

· This takes time, so diabetic must wait before eating a meal.

· If monomers were to be injected a very low concⁿ of solution would be needed – and so a very large volume would have to be injected.

· Two types of insulin are mixed to prepare injection, one which breaks down easily into monomers to give rapid response so a meal can be eaten, and one which breaks down slowly to give low level background insulin for up to 12 hours.

· Usually insulin production responds naturally to food intake – for diabetics food intake must be planned around insulin injections.

· During the 1980’s scientists developed methods of protein engineering that can modify the structure or properties of a protein and it is possible that this can be used to modify insulin so the monomers are stable and do not combine to form hexamers. To do this would need to know;

· Composition and structure of insulin.

· Shape of molecule and which regions form interactions that lead to hexamers being formed.

· The type of intermolecular attractions involved.

· How to modify monomers to prevent these.

EP2 – Protein Building.

Amino acids; the building blocks of proteins.

· See fig 11 page 141 which shows a molecule of insulin.

· The abbreviations in circles represent a-amino acids.

· In insulin there are two short chains of these amino acid residues – (the parts of the original amino acids which are joined together to form the protein).

· There are 20 a-amino acids which combine in various ways to make up all the proteins in the living world. They all have the same basic structure;

H R 0

N C C

H H OH

Note that the NH₂ and COOH groups are both attached to the same crbon atom – the a carbon

Time to look at some functional group chemistry;

· Revise CI 13.3, 13.4 and 13.7

· CI 13.9 will cover amino acids in more detail.

· In each amino acid it is the side chain R which is different and table 1 page 142 shows the R group for each of the 20 amino acids.

· Our bodies contain proteins which must be continually replaced, and this is done from the food we eat.

· The proteins we eat are broken down into their constituent amino acids and then these are assembled in the body into the proteins we require.

· There are 8 essential amino acids, which our bodies cannot synthesise – they must be taken in. The other 12 can be made in the body from carbohydrates and other amino acids.

· Thus we can replace the proteins in our bodies by eating animal proteins or plant material.

· Within our bodies are some proteins which are the same in everybody, but some which are different – for each person the collection of proteins is unique!

· It is the order in which the amino acids are joined – the primary structure – which differentiates between particular proteins.

Now do assignment 1.

Activities EP2.1 and EP 2.2 are two practical exercises.

· In What’s in a Medicine two spectroscopic techniques were covered – IR spectroscopy and Mass spectroscopy. Here we will meet a third technique – nuclear magnetic resonance (n.m.r.) spectroscopy.

CI 6.6 covers the theory of n.m.r.

Activity EP2.3 gives you a chance to use it!

Making Peptides.

· When amino acids join to make proteins it is a condensation reaction with an amide group (peptide link) formed and a molecule of H₂O lost.

· If two molecules of amino acid link this way a dipeptide is formed (see CI 13.9)

· Convention for naming is to start with free NH₂ group on left and read from left to right. Read green box on page 143 and do Assignment 2.

· When chemists react amino acids together they often have to convert the COOH group to a COCl group to make it more reactive.

· Amino acids show another property – optical isomerism –they exist in two forms, the D or L forms depending on the spatial arrangement of the four groups on the a carbon atom. Proteins are built from only the L isomers.

Revise CI 3.3 and 3.5 (shapes of molecules and Isomerism)

CI3.5 will introduce and explain optical isomerism.

· Cells build proteins from L amino acids, and chemists are learning how to use bacteria and yeast cells to manufacture proteins. 3 thing are needed before a protein can be synthesised;

· Knowledge of the primary structure of the protein.

· Supplies of the pure amino acids involved.

· A way of making the amino acid and carboxylic acid groups react more easily.

Now do activities EP2.4, EP2.5 and EP2.6.

How cells make proteins.

· The instructions specifying the primary structure of a protein are carried by molecules of a ribonucleic acid (RNA).

· There are many different types of RNA.

· Messenger RNA’s (mRNA’s) provide the code which tells the cell which amino acids to put together, and in what order, to build a protein.

· Transfer RNA’s (tRNA’s) select and separate the amino acids needed for protein synthesis from those dissolved and mixed together in the cell fluid.

· The cell has a different tRNA for each different amino acid.

· There is also a set of enzymes which recognise the tRNA and its corresponding amino acid. The enzyme catalyses the formation of an ester bond between the COOH of the amino acid and an OH group on the tRNA. The resulting complex then diffuses to the place where the protein is being built.

· RNA molecules have a backbone of ribose sugar molecules and phosphate groups joined together by a condensation reaction.

· One of four bases is attached to each ribose unit. The sequence of bases is different for each RNA molecule. It is the sequence of bases which form the code for protein synthesis.

· Fig 14 page 145 illustrates the structure of a particular RNA molecule, and shows the different ways it can be represented.

· Ribosomes are the cells catalysts for protein production. They contain ribosomal RNA (rRNA) bound to protein molecules.

· When the mRNA and the ribosome have collided in the fluid of the cell, the ribosome moves along the mRNA (like a bead on a chain) reading the code and catalysing the reactions which join the amino acids together in the correct sequence.

Now do assignment 3

Cracking the Code.

· There are only 4 bases so there are not enough for each (or even pairs) of the bases to identify each of the 20 amino acids.

· In fact a triplet code is needed, a combination of 3 bases which tells the cell which amino acid to use. There are then more than enough combinations and some amino acids are defined by more than one triplet of bases.

· These triplet combinations are known as codons and are shown in table 2 page 146.

· Note that there are also codons which stop the protein chain building.

Now do assignment 4

· tRNA molecules recognise and bind to the codons on the mRNA through anti-codons.

· Base G in the anti-codon recognises base C in the codon (and vice-versa)

· Bases U and A are similarly related.

· E.g. Anti-codon CGG will bind to codon GCC (the codon for alanine)

· Remember the codons are on the mRNA and the anti-codons on the tRNA.

Now do assignment 5.

· Study figs 16 and 17 pages 146-147 – these explain the role of mRNA, tRNA and ribosomes in the synthesis of proteins from amino acids. It is very important that you study these carefully and understand the process that is carried out.

· To recognise each other, molecules must have shapes that fit neatly together so that groups can form intermolecular bonds.

· The bases in RNA have flat shapes and fig 18 page 147 shows how the pairs of bases fit together and hydrogen bonds form between them (two in the case of U and A, and three in the case of C and G)

A Permanent Record

· mRNA is destroyed when production of it’s protein is no longer required.

· A permanent record of instructions for protein production is kept in the nucleus of each cell in the form of deoxyribonucleic acid (DNA). DNA and RNA both consist of sugar-phosphate strands with attached bases, but there are important differences (see table 3 page 147);

· The ribose in RNA is replaced by deoxyribose in DNA.

· The base uracil(U) in RNA is replaced by the base thyamine(t) in DNA.

· DNA molecules are normally paired off in sets of two strands in a double helix arrangement. (As proposed in 1953 by Crick and Watson)

· The strands of DNA are held together by hydrogen bonds between the pairs of bases, (T and A, C and G) see figs 19 and 20 page 148.

· The hydrogen bonding is just like that between the codons and anti-codons in RNA.

Now do activities EP2.7 and EP 2.8

· When a cell starts protein production the record in the DNA must be turned into an RNA message carried by mRNA, which is then read to synthesise the protein. Seefig 23 page 149.

· Nearly all cells use the same system;

· DNA codes for RNA

· RNA codes for protein.

· DNA molecules contain information for the production of many mRNA molecules, whereas mRNA molecules contain the code for just one protein.

· A DNA segment responsible for the production of a certain protein is called a gene.The full set of genes in an organism is called it’s genome.

· The human genome consists of about 3.5x10⁹ base pairs. Mapping it has been a vast, international venture since 1988. The sequencing of bases has already been achieved, the next stage is to identify how they are divided into genes and what the resulting proteins actually do. The data is collected using a computer data base.

· Genes are present in the nucleus of cells, but must be ‘switched on’ before protein production starts.

· DNA also contains base sequences to start or stop RNA production, and also ‘junk DNA’ which seems to have no function.

Now do assignment 6, and read ‘DNA fingerprinting’.

EP3 – Genetic engineering.

Changing genes.

The technology for using yeast and bacteria cells to produce useful chemicals has been around for a long time. Brewers have used yeast to convert sugar into alcohol for thousands of years and more recently pharmaceuticals like penicillin have been made in fermenters using moulds.

Even more recently scientists have learned how to build human genes into bacterial or yeast cells, which can then be used to generate proteins. Fig 29 page 151 represents a generalised approach of the use of bacterial cells to produce the hormone insulin, needed for diabetics. This is an example of genetic engineering, or recombinant DNA technology.

Examples of Genetic engineering;

· Making pure samples of human proteins e.g. Insulin, human growth hormone or factor 8 (a blood clotting agent). Genetically engineered factor 8 reduces the risk of haemophiliacs contracting AIDS, hepatitis, CJD etc. from infected donors. A single genetically engineered tobacco plant can produce as much factor 8 as a 1000dm³ fermentation vessel.

· If the protein coat of a virus – minus its dangerous contents! – is genetically engineered and injected into the body it can act as a vaccine. (hepatitis B vaccine is produced this way)

· A new oil digesting ‘superbug’ has been created using a collection of genes from several bacteria.

· PHA polymers are made by bacteria and are biodegradable (see DP storyline).

· The treatment of cystic fibrosis. ‘Healthy’ copies of the gene for producing the protein necessary for allowing passage of chloride ions and their associated water molecules across membranes of the lungs, thus keeping them moist and clean, are introduced directly into the air passages of sufferers. They can produce this protein themselves.

· Production of plants with modified properties e.g.

· More flavoursome tomatoes.

· Inbuilt resistance to certain pests. E.g. Maize plants are prone to the corn borer. The bacterium bacillus thuringiensis produces an a-endotoxin protein which is deadly to this pest. The plants can be sprayed with the endotoxin, but genetic engineering should allow the maize plants to actually produce it themselves and give them built in protection.

· Plants which can produce certain pharmaceuticals drugs or polymers.

· Tomatoes which deteriorate more slowly after picking so they can be left on the vine longer to develop their own natural flavour.

· Plants which can tolerate salt and still grow well.

· There are of course environmental and moral issues to be considered, especially with crops where it is difficult to contain experimental work, how do you stop bees for instance going in a particular field of genetically modified crops? (assignment 7 will make you think about these.)

SL EP4 – Proteins in 3D

Folded chains.

Cow, pig and human insulin can all be used to treat diabetes, even though they have different primary structures. Insulin, like most proteins, has a precise shape which arises from the folding together of the chains. The action of insulin is critically dependant on this shape, and as long as different molecules fold to the same shape they may have the same action. Chain folding gives proteins their 3 dimensional shape, and places chemical groupings into positions where they can interact most effectively. There are four types of interactions important in folding;

1) Instantaneous dipole-induced dipole attractive forces – between non-polar side chains on amino acids. Tends to be in centre of protein molecule so non-polar groups do not interfere with H-bonding with surrounding water molecules.

2) H bonding – between side chains such as –CH₂OH in serine and –CH₂CONH₂ in asparagine, between peptide groups which link the chains together and between groups on the outside of the protein molecule and surrounding water molecules.

3) Ionic attractions – between ionisable side chains e.g. –CH₂COO^- in aspartic acid and –CH₂ CH₂ CH₂ CH₂NH₃⁺ in lysine.

4) Covalent bonding – The SH groups on neighbouring cyestine residues can be oxidised to form –S-S- links. There are three such links in human insulin – see fig11 page 141.

Now revise intermolecular forces – CI5.3 and CI5.4

The structure of a molecule of protein can be described in 3 stages;

Primary structure – The order of amino acid residues.

Secondary structure – The way in which the chains are folded or twisted in a regular manner due to hydrogen bonding. Two arrangements are common;

· Tightly coiled into a helix where the C=O group on one peptide link forms a H-bond to an N-H group four peptide links along the chain. See fig34a page 154.

· Stretched out into regions of extended chain, which lie along side each other and H-bond to form a sheet. See fig34b page 154.

Tertiary Structure – The chains may then fold up further, the overall shape being stabilised by the four types of interactions mentioned earlier. Figs 35 and 36 page 154 show the tertiary structure of the enzyme gyrase and the hormone insulin as ribbon diagrams. It is interesting to compare fig 36 with the earlier representation of insulin – fig 11 – which simply showed the primary strucyure.

Insulin Hexamers.

· Insulin monomers stick together to form dimers and hexamers, and this is also due to intermolecular attractions. Fig 37 page 155 shows a space filling model of one insulin monomer – which shows that one side of the molecule consists mainly of amino acids with non-polar side chains (the white spheres)

· These non-polar groups, if left on the outside of the molecule would disrupt hydrogen bonding to surrounding water molecules.

· For this reason the monomers form dimers where the two non-polar sides of the molecules come together – to reduce this disruption.

· The formation of the hexamer then brings more non-polar groups together and leaves an outside surface which consists mainly of polar groups which can then hydrogen bond with water strongly. See figs 38, 39 and 40 on page 155.

· The joining of insulin monomers into hexamers is referred to as its quaternary structure.

SLEP5 – Giving Evolution a push.

Insulins that stay single.

· Injecting insulin monomers would be better for diabetics as they work faster.

· The hexamer and its monomers are in dynamic equilibrium;

Insulin hexamer 3 Insulin dimers 6 Insulin monomers

Ins₆ 3Ins₂ 6Ins

· The position of equilibrium depends on the concⁿ of the solution.

· As hexamers spread from injection site and become more dilute the equilibrium shifts to the right.

· Insulin is mainly in form of monomers when diluted to 1x10^-8 mol dm^-3 but injecting a solⁿ of this low conⁿ would be impractical as too large a volume would be required.

· One idea was to modify the structure of the insulin molecules to prevent them sticking together, this would move the position of equilibrium to the right so the monomers would be stable in more concentrated solutions.

· Fig 43 page 157 shows an insulin dimer. There are two ionised –COO^- groups on glutamic acid residues (the red spheres) in the B13 position (amino acid 13 in the B chain) and two polar –CH₂OH groups from serine residues (the blue spheres) in the B9 position, which are close together. Attractive forces between these groups would help keep the dimer together.

· If the serine residues at B9 could be replaced with aspartic acid residues there would be 4 –COO^- groups close together and this would push the monomers apart.

· Scientists study changes like this using computer models before any experiments are done, and information from X-ray diffraction studies provides a lot of information about the stucture of molecules to make this possible. See fig 44 page 157.

Designer genes.

· To manufacture protein you don’t have to string all 51 (in the case of insulin) amino acids together. Because of the remarkable ability of molecules to ‘recognise’ each other you only have to build part of the molecule – the cell will do the rest!!

· In the case of insulin the codon for serine (AGG) at position B9 needed to be changed for the codon for aspartic acid (CTG) so only the six amino acids around this position needed to be synthesised. See fig 45 page157. Remember DNA has two strands, the gene can be inserted into a plasmid of DNA in a bacterial cell and then certain viruses will ‘unzip’ the two strands.

Step 1. Synthesise the section of DNA required i.e. in this case the 18 bases of strand 1, and introduce this into the bacterial cell.

GAA ACA CCC AGG GTG GAA

Step 2. In the cell this will act as chemical ‘magnet’ and stick to the complementary section of strand 2, this is because T sticks to A and C sticks to G, and there is little or no chance of the same sequence of bases appearing anywhere else in the plasmid,

CTT TGT GGG TCC CAC CTT Strand 2

GAA ACA CCC AGG GTG GAA

STEP 3. The normal cell processes in the bacteria will then recreate the plasmid double helix.

CTT TGT GGG TCC CAC CTT Strand 2

GAA ACA CCC AGG GTG GAA Strand 1

Step 4. The chances of finding the same sequence of 18 bases elsewhere in the plasmid are so small that the cell will tolerate a mistake. If scientists replace the highlighted AGG with CTG (codon for aspartic acid) it will stick onto the same place, even though two bases don’t match, and then complete the double helix as normal.

Strand 2

Strand 1 CTT TGT GGG TCC CAC CTT

carrying GAA ACA CCC CTG GTG GAA

‘mistake’ CTG region where bases on

codon for Asp. diff strands can’t interact.

Step 5. When the bacteria split to multiply a new strain of bacteria will be produced which will carry the gene for monomeric insulin.

Original plasmid carrying New type of plasmid carrying

Codon for Ser at B9 codon for Asp at B9

CTT TGT GGG TCC CAC CTT CTT TGT GGG GAC CAC CTT

GAA ACA CCC AGG GTG GAA GAA ACA CCC CTG GTG GAA

Treating diabetics.

· Monomeric insulin has been used to treat diabetics and its release into the blood stream more closely resembles that of natural insulin production in non-diabetics. This can be seen by comparing fig 48 page 158 and fig 6 on page 139.

· The monomeric insulin gets into the blood stream much more quickly because it does not need to break down from hexamers.

· This gives diabetics more control as they can have the injection just before they eat, so they no longer have to predict when food will be available.

· Read ‘A Breakthrough’ to find out how the small molecule phenol can also help to make insulin less likely to join up into dimers and hexamers.

SLEP6 – Enzymes.

You can buy reagent strips from a pharmacist which will determine the presence of glucose in the urine. This can be used as a test for diabetes as when levels of glucose in the blood reach a certain level it ‘spills over’ into the urine. The green box on page 159 explains how the strips work – they rely on 2 enzymes, an indicator and a buffer – and this illustrates four important points about enzymes, that they are;

· Catalysts

· Highly specific to certain reactions

· Sensitive to pH

· Sensitive to temperature

Active sites.

· Enzymes often have a precise tertiary structure which matches the structure of the substrate (the molecule which is reacting). They then react together in a lock and key mechanism – see fig 52 page 160.

· See fig 51 page 160 – the active site on the enzyme lysozyme is very clearly seen.

· Within the active site are chemical groups, e.g. the side chains on amino acid residues, which bind the substrate and may react with it.

· The bonds have to be weak so they can be easily reversed when the products leave the active site, usually H bonds or interactions between ionic groups.

· The binding may cause other bonds within the substrate to weaken, or may alter the shape of the substrate to bring other parts into contact to help them react.

· After the reaction the products leave the enzyme – which goes on to react again. E + S ES ® EP ® E + P

· In reality it may not be this simple!!

Enzymes as catalysts.

· Catalysts provide an alternative pathway for a reaction with a lower activation enthalpy this is illustrated in fig53 page 161.

· When the activation enthalpy is lower a reaction will take place more quickly.

· Remember from CI 10.3 that if substrate concentration is high then all the active sites on the enzyme will be occupied (enzymes only tend to be present in trace amounts. Increasing the concⁿ of the substrate will have no further effect on the rate of reaction – the reaction is zero order with respect to substrate.

· If substrate concⁿ is low then not all the active sites will be occupied so increasing the concⁿ of S will increase the rate of reaction (twice as many S molecules mean twice as fast a reaction) i.e. the reaction is first order with respect to substrate.

· If an active site contains an ionisable group it will be affected by pH. E.g. if a –COOH group is present, which acts as a H⁺ donor in the reaction, then raising the pH (adding e.g. OH^-) will make it unable to work.

· Changing the tertiary structure (the shape) of an enzyme will destroy the active site and denature the enzyme. Fig 54 page 161 illustrates this. One way of doing this is by raising the temp, as the tertiary structure is held together by weak dipole-dipole and H bonds, which are broken as they vibrate more vigorously if temp is raised.

· Thus enzymes are very sensitive to changes in pH and temperature.

Enzymes can be used in the following applications;

· Diabetes strips or other medical diagnostic kits.

· Biological washing powders contain enzymes e.g. protease to digest protein, lipases to digest fat stains, and cellulases to break down surface fibres and prevent that ‘fluffy’ look. New technology enables them to be used at higher temps or be more effective at lower temps.

· Starch is broken down by a amylase to produce glucose syrup to sweeten food.

· Cheese is made using rennet enzymes to break down the milk protein casein.

· Waste treatment e.g. removal of cyanide ions after gold extraction, or to break up oil spillages.

· Baking, brewing and fruit processing.

· To give a ‘stonewash’ appearance to denim.