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Introduction

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

This unit examines how self-assembled structures based on lipids and proteins provide a framework for cellular processes.

This unit is an adapted extract from the Open University course Engineering small worlds: micro and nano technologies (T356).

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Learning outcomes

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

After you have completed this unit you should be able to:

describe and give examples of how self-assembly enables construction ‘from the bottom up’ in natural materials;
explain what is meant by primary and higher-order structure in proteins and give examples;
give examples of the range of functions carried out by proteins within cells;
describe how a combination of strong and weak bonding within biopolymers and lipids is used to build hierarchical structures with common structural elements and finely tuned properties, including calculations where appropriate;
explain how both positive and negative design principles must be applied to the design of molecular devices and comment on the challenges involved in attempting such design.

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1 Biological materials

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

Materials engineers have long recognised the impressive range and combination of properties offered by biological materials. Figure 1 shows some representative examples of the combination of tensile strength and toughness (measured by Young's modulus, or elastic modulus for polymers) offered by natural materials, with some more common engineering materials included for comparison.

I'm using the term ‘biological material’ here to describe materials of natural origin. The more general term ‘biomaterial’ can include both natural and synthetic materials that are used to replace or interact with part of a living system.

Figure 1 Tensile strength and toughness (Young's modulus, or elastic modulus for polymers) of a range of natural materials with some common engineering materials included for comparison. The (diagonal) shading indicates the energy to break: the darker the shading, the higher the value – note the logarithmic axes

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Many biological materials approach or exceed the strength of steel: in fact, some forms of spider silk exceed it by a good margin. If density is taken into account, the specific strengths (strength per unit mass) are even more impressive. Biological materials frequently combine high strength with relatively low stiffness, and this leads to high values for the energy needed to break the material, as indicated by the shading on the diagram. You will see that collagen, the main component of skin and tendons, is particularly impressive in this respect. Biomaterials tend to have a specific combination of properties, closely suited to their function, which can be difficult to match with manufactured alternatives.

If we look closely at natural materials we find that their properties are controlled right down to the molecular level, with each material uniquely adapted to suit a particular purpose and to interact in complex ways with its neighbours in response to changes in circumstances. Consider bone, for example. Natural bone is a composite, combining polymeric protein chains with crystals of ceramic calcium phosphate. In the body it responds to stress levels, being absorbed when low stress levels indicate that it is not needed, growing thicker when the stress levels are high and receding in situations where the stress levels are so high that damage could occur. Given this intelligent response, together with the mismatch in mechanical properties of bone and steel evident from Figure 1, it is no wonder that the design of effective hip replacements, for example, is so technologically challenging.

One of the reasons that it is so difficult to match the properties of biological materials is that they are built within cells from the bottom up, unit by unit, to a strict specification. If, by studying their structure and method of production, we can learn to copy the design principles used in nature, then the scope for producing new, smarter materials outside the cell is immense.

SAQ 1

A particular spider of mass 50 mg can spin dragline silk with a tensile strength of 1.2 GPa ± 0.2 GPa. This compares to 0.55 GPa ± 0.05 GPa for a fibre of nylon 6 (a common form of nylon). The tensile strength is the force per unit area needed to break the fibre.

(a) What diameter must the dragline exceed to ensure that it can support the weight of the spider?
(b) What diameter fibre would be necessary to support a 60 kg human? What is the comparable value for nylon 6?
(c) Spider silk and nylon 6 have similar densities of about 1200 kg m⁻³, while that of steel is 7800 kg m⁻³. Assuming the steel to have the same tensile breaking stress as the spider silk, calculate the mass per metre of a fibre of sufficient diameter to support the 60 kg human, for all three materials.

Answer

(a) Use the bottom-of-the-range value of the tensile strength to find the minimum diameter, i.e. 1.0 GPa for dragline silk, 0.5 GPa for nylon.

We know that the tensile strength is the force per unit area needed to break the fibre; thus:

where r is the minimum radius, in m.

Therefore, at breaking point:

The minimum diameter of the dragline that will support the weight of the spider is 0.78 µm.

(b) Using the same method as in (a), but with the weight equal to (60 × 9.8) N, gives values for the minimum diameter required to support a 60 kg human of 0.86 mm for dragline silk and 1.2 mm for nylon 6.

The volume of a metre of fibre of radius r is equal to . For dragline silk, using the value for r calculated in part (b):

The equivalent value for nylon 6 is 1.4 g m⁻¹, and for steel 4.5 g m⁻¹

Several of the materials in Figure 1, including collagen, keratin and silk, are proteins. Proteins fulfil many important roles in cells and make up much of their mass. You probably think of proteins in terms of foodstuffs like eggs, fish and meat; in fact, looking at the contents of the food that we eat gives a pretty good guide to the range of materials found in cells. For example, a chicken curry from my fridge lists the following composition (by mass): 15% protein, 9% fat, 5% carbohydrate, 0.8% salt (and the rest mainly water). Compare this to the chemical composition of a typical mammalian cell (shown in Table 1) and you'll see that they are remarkably similar.

Table 1 Chemical composition of a typical mammalian cell

Compound	Mass / % of total
Water	70
Proteins	18
Lipids (fats and oils)	5
Polysaccharides (carbohydrates)	2
Nucleic acids	1
Small molecules and ions	4

Despite their complexity, living cells are constructed almost entirely from four basic classes of chemical compound:

lipids, which are used for energy storage and to form cell membranes
proteins, which fulfil a very wide range of functions from structural materials to nanoscale motors
sugars and polysaccharides, which are important for energy storage and as structural materials in plants
nucleic acids, DNA and RNA, which although less prolific are hugely important as they allow information to be stored and read within cells.

In this unit, I'll introduce lipids and proteins as the materials that give structure to cells.

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2 Construction with lipids

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

The cell membrane is constructed from lipids. Chemically, lipids are a rather varied group of compounds that include all the substances you might already think of as fats or oils. What they have in common is that they are all insoluble in polar liquids like water, but soluble in organic (carbon-based) solvents: by this I mean the sort of smelly solvents you tend to find in paints, glues and degreasing agents; chloroform is one example. Lipids make up the fatty components of living organisms and are the major components of margarine, cooking oil, butter and the white fat associated with meat. The terms ‘fat’ and ‘oil’ simply describe whether a lipid is solid (a fat) or liquid (an oil) at room temperature. Fats and oils are important in cells as energy storage compounds, while the cell wall itself is constructed from a class of lipids known as phospholipids.

The reason for their insolubility in water is that lipids contain large hydrophobic groups. Hydrophobic literally means ‘water-hating’ and generally applies to non-polar substances or chemical groups. In contrast, polar substances or groups are termed hydrophilic or ‘water-loving’. As the name suggests, they have an affinity for water and are more likely to dissolve in it.

The interplay between hydrophilic and hydrophobic groups is important in many biological materials.

Their hydrophobic nature means that lipids tend to separate from water and from all water-soluble, hydrophilic compounds and to group themselves together as large hydrophobic aggregates, held together by weak hydrophobic interactions. This is why oil and water don't mix. Because of the weak bonding between the molecules, lipids tend to be fluid aggregates that can easily change shape. Think of a lump of lard: if you press on it, it dents. A material like this with a certain amount of fluidity can be extremely useful in certain situations. Insolubility in water is also a useful property when it comes to forming a barrier to keep some substances separate from others.

Many important lipids, including the phospholipids that make up cell membranes, contain both hydrophobic and hydrophilic groups. Such molecules are termed ‘amphiphilic’ and exhibit particularly interesting properties when mixed with water. Figure 2 is a sketch representing an amphiphilic lipid molecule, such as a phospholipid. The two ‘tails’ are based on chains of carbon atoms and are hydrophobic. The head is a polar hydrophilic group. Other lipids may have a different number of tails.

Figure 2 Simple representation of a lipid molecule. This one has two hydrophobic (water-hating) tails and a hydrophilic (water-loving) head, like a phospholipid

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Consider how amphiphilic lipid molecules might aggregate together in a strongly hydrophilic, watery environment. The hydrophobic chains will avoid water, coming together and interacting with one another on the inside of the aggregate. This leaves the hydrophilic head groups on the outside of the aggregate, in contact with water molecules. The charged head groups will repel each other to some extent, so the arrangement they adopt must achieve a balance between the attractive forces (between the heads and the surrounding water) and the repulsive forces (between neighbouring heads). A wide range of structures can be produced, depending on the shape of the molecule, the nature of the head group, and the concentration of the mixture. Some examples are shown in Figure 3.

Figure 3 Cross sections of some of the common structures adopted by amphiphilic lipids in water: (a) micelle; (b) inverted micelles; (c) bilayer; (d) bilayer vesicle, or liposome

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The formation of well-defined structures by amphiphilic lipids is the first of several examples we will see of self-assembly, which is a key feature of many natural systems. Self-assembly means that no outside influence is needed to cause the molecules to organise in a particular way: it is a direct consequence of their molecular design and the electrostatic forces that exist, on very small scales, between individual molecules. It relies on Brownian motion to shake the molecules around so that the optimum arrangement can be found.

Some authors would describe the behaviour of lipids as self-organisation, since there is more than one possible way of arranging the molecules, and reserve the term self-assembly for more specific interactions such as those found in proteins. Here, I will not differentiate between the two and only the term self-assembly will be used.

Amphiphilic lipids, which contain a polar, water-soluble group attached to a non-polar, water-insoluble hydrocarbon chain, have many uses. They are widely used as surfactants (substances that reduce the surface tension of a liquid) and emulsifiers (substances that promote the formation of a stable mixture of oil and water), and you'll find plenty of examples around the home. Soaps and detergents work by forming micelles when added to water; see Figure 3(a). These micelles are able to surround non-polar contaminants such as grease, encapsulating them within their hydrophobic core and thus allowing them to dissolve in water, as illustrated in Figure 4. The charged heads on the surface ensure that the micelles repel one another, preventing them from clumping together – effectively emulsifying the dirt. In other circumstances (higher concentrations, or differently shaped lipids) the micelles may be inverted, as in Figure 3(b). In this case the hydrophilic heads point inwards, surrounding small globules of water within a matrix of lipids.

Lipids are also common in the kitchen. Many foodstuffs contain both oil and water, and a variety of emulsifiers are used to stabilise their structures. For example, lipids found in egg yolk act as an emulsifier in egg mayonnaise, enabling a stable emulsion to be formed from oil and vinegar.

Figure 4 Dirt surrounded and made soluble by lipids

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The double-layered lipid structure shown in Figure 3(c) illustrates the basic arrangement of the phospholipids in a cell membrane. A more detailed view is given in Figure 5. (Plant cells possess a tough outer cell wall in addition to the phospholipid membrane. Here we will concentrate only on the membrane itself.) Within the membrane the phospholipids cluster together to form a sheet-like structure, with all the hydrophobic tails in the centre, screened from the surrounding water. The phospholipid bilayer of a cell membrane is a very effective barrier, preventing hydrophilic molecules from passing freely into, or out of, the cell. Membranes also define the boundaries of structures within cells, restricting various cellular functions to particular compartments of the cell. Another important function of the membrane is to support other molecules, which allow the cell to interact with its neighbours and with the external environment. Larger molecules, such as proteins, ‘float’ within the fluid lipid bilayer; we'll return to consider these later.

Figure 5 A biological membrane; a typical membrane thickness is about 10 nm. The green structures are membrane proteins; some of these traverse the membrane and others are on its surface

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The final structure illustrated in Figure 3 shows a lipid bilayer bent round on itself to form an enclosed compartment. These structures may be described as liposomes or vesicles, depending on the context. (The term liposome tends to be used for synthetically produced bilayer structures. The term vesicle is used more generally, but has a specific meaning within cells as a small spherical bilayer sac in which substances are transported or stored.) The bubbles formed by soaps and detergents also have a bilayer structure similar to that in Figure 3(d) and provide us with a reasonable model for a cell membrane. Blowing a bubble shows us how a flexible membrane can be bent round on itself to form an enclosed space; you can demonstrate the fluid and self-sealing nature of a membrane by moving an object, such as a pencil, through a soap film and then removing it.

Liposomes can be used to encapsulate water-soluble substances such as vaccines, drugs, enzymes or vitamins and deliver them to different cells in the body. Drug-delivery systems based on lipids are under development and show considerable potential. Elsewhere, aggregates of lipids in solution are being used to produce nanoparticles of precisely controlled size and shape, such as the quantum dots available for molecular labelling. Langmuir-Blodgett films (named after the scientists who first studied them) provide a well-established method of generating structures built up from lipid mono layers that have potential for electronic applications.

Box 1 Langmuir-Blodgett films

A Langmuir-Blodgett (LB) film is made up of highly organised layers of lipids, each just one molecule thick, deposited onto a solid substrate by repeatedly dipping it into a water bath with a lipid film floating on top. By varying the experimental conditions and the lipid used, multilayer stacks can be built up with precisely controlled molecular compositions. LB films may consist of a single layer or many, up to a depth of several micrometres, and can be made with various electrochemical and photochemical properties. They are being investigated as possible structures for tiny integrated circuits.

Synthetic bilayer sacs like those in Figure 3(d) are relatively easy to make and provide a reasonable model for a (very!) simple cell. But for many nanoengineering applications it is much more useful to have a lipid layer that is tethered in a fixed position. Fixed bilayer membranes supported on solid surfaces are much more stable and often offer a more promising route for the development of structures that mimic nature.

In related developments, the self-assembling properties of lipid-like molecules containing thiol groups have been used in microcontact printing and dip-pen lithography to produce self-assembled features on gold surfaces at scales as small as tens of nanometres. (Thiol groups, which contain sulphur and hydrogen, are particularly useful for ‘sticking’ biological molecules to metal surfaces.) By varying the chemical structure of the molecules used, the surface chemistry can be modified so that the thiol may function as an etch resist or produce a surface with markedly different properties in different areas.

SAQ 2

(a) What do the four structures in Figure 3 have in common?
(b) If salt (sodium chloride, Na⁺Cl⁻) were added to the mixture in Figure 3(a), where would it go? What about petrol (which contains hydrocarbon chains, such as C₈H₁₈)?

Answer

(a) In all the structures the hydrophilic heads of the lipids are in contact with the water, while the hydrophobic tails are not.
(b) The salt would dissolve in the water surrounding the micelle. Petrol is hydrophobic, and would mix with the lipid chains in the centre of the micelle.

SAQ 3

Suggest which of the structures shown in Figure 3 would be suitable for the following applications:

(a) restricting the size of crystals growing from solution in water
(b) suspending a hydrophobic pigment molecule in a water-based paint
(c) providing a boundary between an area of high concentration and an area of low concentration within a liquid
(d) delivering a water-soluble vitamin to a cell.

Answer

(a) For this we need an enclosed compartment within which the crystals can grow. Inverted micelles would provide this, and the size of the cavity can be varied by choosing different lipids. Although liposomes also provide compartments, crystals of other sizes could grow from the surrounding liquid.
(b) Micelles are able to surround the hydrophobic molecules and render them soluble.
(c) The bilayer would be most suitable here; concentration gradients across lipid bilayers are very common in cells.
(d) Here a liposome/vesicle-based delivery system could be used, with the vitamin enclosed within the lipid compartment.

The principles that govern the self-assembly of lipids can also be applied if we take small units with different chemical characteristics and join them together. This is the situation that exists in a block copolymer, as described in Box 2 Self-assembly in block copolymers.

Box 2 Self-assembly in block copolymers

Block copolymers have chains made up of alternating sequences of two or more different repeating units. Let's assume that there are just two components, which I'll call A and B, so that a section of chain might look something like this:

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Now suppose that the A units are hydrophilic and the B units are hydrophobic. The As will try to separate from the Bs and clump together, and vice versa, leading to phase separation on a tiny scale. The structures that can be produced are very similar to those obtained from lipids but, in this case, the result is a solid, with greater connectivity throughout the structure because the units are linked together in chains. Figure 6 shows the sort of structural arrangements that can be obtained. By changing the length and proportions of the different blocks, the microstructure, and hence the bulk properties of the material, can be finely controlled.

Figure 6 Examples of the different micro structures adopted by block copolymers with hydrophobic and hydrophilic elements

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3.1 Protein diversity

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

Of course, our bodies can't just be made up of squidgy bubbles of phospholipid, or we would collapse in a heap on the floor! Stiffer frameworks, both inside and outside the cells, also exist and help to define shape and add strength. These frameworks are formed largely from structural proteins, a class of polymeric materials that form fibres and filaments to provide mechanical support for cells and tissues. Structural proteins are made inside cells but are often then moved into the space surrounding the cells, where they interact together to form a three-dimensional polymer network, permeated by fluid.

Section 2 introduced the idea of self-assembly in a simple system, but the real masterpieces of self-assembly are the proteins. As well as providing structure, proteins play a central role in defining the function of different cells. There are many of them – around 10 000 types in a typical cell – and they fulfil a wide range of functions. Some examples are listed in Table 2.

Table 2 Examples of the functions fulfilled by proteins within cells

Type of protein	Function	Examples
Structural proteins	provide mechanical support for cells and tissues	skin and tendons contain collagen
		hair, nails, beaks and feathers are all based on keratin
		actin forms part of the skeleton within cells
Enzymes	biological catalysts, which speed up essential chemical reactions	many enzymes are involved in digestion, or breaking down toxins, e.g. pepsin, alcohol dehydrogenase
		DNA polymerase enables DNA to be made
		ATP synthase converts ADP to ATP
Transport proteins	transport small molecules or ions from one place to another	haemoglobin carries oxygen around in the bloodstream
		membrane transporters carry molecules or ions across the cell membrane
Signalling proteins	carry signals between or within cells	hormones: for example, insulin, which controls blood sugar levels
Receptor proteins	detect signals and transmit them within cells	rhodopsin detects light in the eye
		insulin receptor binds insulin
Motor proteins	generate movement in cells	myosin is involved in muscle movement
		kinesin carries cargo around cells

Proteins are also used for storage of essential nutrients, for labelling things so that they can be recognised by other cell components, for sending instructions to DNA, and for many other specialised functions.

The action of a protein is intimately linked with the three-dimensional shape that it adopts, although it is seldom possible to guess the function of a protein from its shape alone. Some examples of protein shapes are shown in Figure 7.

Figure 7 A selection of proteins, based on structures from the Protein Data Bank. In this figure a ‘space-filling’ representation has been used to emphasise the overall shape of the molecules

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Structural proteins tend to be long and thin, like the strands in a rope. The molecules they are based on may be stretched out to form fibres, like the collagen in Figure 7 (a), or built up from individual units in which the chain is coiled up rather like a ball of string: the actin in Figure 7 (b) is an example, with an individual unit shown at the top of the diagram. However, most proteins have a more globular shape. In general, the globular proteins have more specialised roles than the structural proteins.

Enzymes are proteins that act as catalysts, helping to promote certain chemical reactions. In some respects all proteins can be regarded as enzymes, since they provide the means for a particular reaction to take place; however, the term tends to be reserved for those proteins whose associated chemical reaction is the primary goal of the process and not just a step on the way towards, for instance, movement. Pepsin, Figure 7(c), is an enzyme involved in breaking down food during digestion. Alcohol dehydrogenase, Figure 7 (d), is an enzyme that many of us should be grateful to: it is the body's primary defence against alcohol, a toxic chemical that compromises the function of our nervous system. This enzyme catalyses the removal of hydrogen (hence the name dehydrogenase) from alcohol, which is the first step towards breaking it down into harmless substances. The high levels of alcohol dehydrogenase in our liver and stomach detoxify about one stiff drink each hour, slow enough for us to enjoy the effects of our folly but fast enough – we hope – to avoid permanent damage. You can read a little more about how enzymes work in Box 3 Biological catalysts.

Figure 7 also shows examples of molecular pores and channels that let certain substances cross the cell membrane. The porin protein in Figure 7 (e) has three permanently open pores, while the potassium channel in Figure 7 (f) possesses a single channel that opens and closes in response to signals and is specially designed to allow only potassium ions through. In the figure, both of these are drawn as if looking down on the membrane; a potassium ion is shown in green in the centre of Figure 7 (f). Sometimes, the process of getting ions across a membrane is helped along by mechanical pumping. The calcium pump shown in Figure 7 (g), this time as a cross section through the lipid bilayer, is essential for maintaining a difference in calcium ion concentration across a cell membrane.

The insulin in Figure 7 (h) and the G-protein in Figure 7 (i) are both signalling molecules, one simple and one more complex. The message carried by insulin is passed on when it fits into a suitably shaped notch on a receptor protein within a cell membrane, while the G-protein is one of a class of proteins that carry messages into the cell itself. The two little ‘ears’ at the top of the G-protein are in fact lipid chains, which insert themselves into the lipid membrane inside the cell and keep the protein tethered there, where it is needed.

The tiny octopus-like structure called prefoldin, shown in Figure 7 (j), helps newly synthesised proteins to fold into the right shape. The restriction enzyme, Figure 7 (k), and DNA polymerase, Figure 7 (l), are both involved in DNA processing, and are shown attached to a portion of a DNA chain (coloured in pink and green). Finally, the myosin illustrated in Figure 7 (m) is a molecular motor involved in muscle contraction..

Box 3 Biological catalysts

Enzymes are biological catalysts that work by providing a low-energy pathway for a reaction, allowing it to happen more readily than would otherwise be the case. As with most proteins, it is the three-dimensional shape adopted by the enzyme protein that makes it suitable for its specific function. Enzymes have binding sites on their surface where particular molecules (ligands) can fit. In this case, the specific binding site is usually known as the active site, and the molecule that can fit into it is called the substrate. Each enzyme is precisely tailored to catalyse a specific reaction: the process is illustrated in Figure 8.

The substrate is not just a suitable shape to fit into the cavity of the active site: it also has a charge distribution that allows weak bonds to form between the two. Interactions between the enzyme and the substrate allow a chemical reaction to happen more easily, by lowering the energy barrier that has to be overcome for the reaction to take place. In many cases this is because bonds in the substrate become slightly distorted when it binds to the enzyme. Strained bonds are easier to break, so the energy required to break the substrate down into the reaction products is lower. Finally, the reaction products are released, leaving the enzyme intact to catalyse further reactions.

Enzymes are widely used in brewing and food production; they also have other domestic uses. For instance, ‘biological’ washing powders contain enzymes to help break down stubborn stains, in much the same way as food is broken down in our stomachs. These are examples in which naturally occurring enzymes are used for pretty much the same purpose as nature designed them. Nanotechnologists are now able to create new biological catalysts by ‘tweaking’ existing enzymes – for instance, to enable them to work over a wider range of temperature or acidity, or to increase their efficiency – or even by using genetic engineering to create new enzymes.

Figure 8 Enzyme action: catalysing a reaction, (a) The substrate is the right shape to fit into the active site; (b) it bonds there, causing it to distort and thus lowering the energy barrier; (c) this allows it to break down into products, which then leave the active site

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SAQ 4

State six major functions fulfilled by proteins in cells.

Answer

Six major functions fulfilled by proteins in cells are providing structural components, acting as catalysts, moving things around, carrying signals, receiving and passing on signals, and generating movement. You could also have mentioned storage of essential nutrients, labelling things for recognition, and sending instructions to DNA.

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3.2 Protein structure

The Open University — Thu, 30 Jun 2011 08:32:47 GMT

Proteins are one example of a biopolymer. You will already be familiar with synthetic polymers such as polyethylene and nylon: long chains made up of many thousands of repeating units, called monomers, linked together by strong covalent bonds. Polymers are particularly versatile materials because of the very different strengths of the bonds between monomer units in the chain (strong) and between one chain and another (weak). By varying the arrangement of chains within a material a huge range of properties can be achieved, from extended chain fibres as strong as steel, to soft rubbery networks where the chains are randomly coiled together, or rigid networks where cross-linking keeps every chain tightly pinned in place. The same principles apply to biopolymers, although the chemical composition of the chains tends to be rather more complex.

Despite the huge number and diversity of proteins in cells, they are all based on the same relatively small number of fundamental building blocks: 20 different amino acids. Amino acids are a class of compound with the same basic chemical structure, Figure 9 offers a diagrammatic representation and Figure 10 shows the chemical structure.

At the centre of each amino acid is a carbon atom, which forms bonds to four other groups. Three of these groups are always the same. Two of them can react together to link individual units into a chain, while the third common group is a hydrogen atom. What distinguishes one amino acid from another is the fourth group, known as the R group, shown as a hook in Figure 9. There are many possibilities for R, but only 20 of them are found in proteins in nature, at least on this planet. Figure 11 shows a few amino acid units linked together, each with a different R unit, and Figure 12 shows the chemical bonds involved.

Figure 9 Diagrammatic representation of an amino acid

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Figure 10 Basic chemical structure of an amino acid

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Figure 11 Diagrammatic representation of four different amino acids linked together

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Figure 12 Chemical structure of a protein chain

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The amino acid units are linked together along the chain by strong covalent bonds, which I have represented in Figure 11 by tightly interlocking pieces. The bonds formed are exactly the same as those that link the units together in synthetic nylons, although (for no particularly good reason) the terminology used is different. In nylons, the monomers are usually referred to as amides, the polymer as a polyamide, and the bonds linking the monomer units together as amide bonds. Biochemists are more likely to refer to amino acids, polypeptides (the protein chains) and peptide bonds.

Why have I represented some of the R units by hooks? As shown in Figure 11, the R units protrude from the side of the polypeptide chain. Some of them are very small (e.g. a hydrogen atom) while some are much larger. The side groups play an important role in the behaviour of a protein because they can form weak bonds with other units, and help to stabilise particular folded-chain arrangements. The different hooks are intended to show that the side units can link together in different ways, but that these links are much weaker than the links along the chain.

Protein chains may be many hundreds of units long, so the number of possible sequences is enormous. The thousands of different proteins found in cells represent just a small fraction of these possibilities; they have evolved over time to perform specific biological functions. Some amino acids may not be used at all in a particular protein, whereas others may occur many times. Each different protein is unique because it has its own individual sequence of amino acids along its length. The sequence of amino acids that makes up a particular protein chain is known as the primary structure of the protein.

Biochemists use the terms ‘protein’ and ‘polypeptide’ rather loosely and interchangeably, and both terms will be used here as well. When a protein is synthesised in a cell, the linear polypeptide chain folds as it is produced. The biological activity of this newly formed molecule depends on its three-dimensional, folded structure, and when considering a polypeptide chain as a three-dimensional structure, it is generally referred to as a protein.

The sequence of amino acids along a polypeptide chain determines the primary structure of the protein, but the chain is flexible and has the potential to fold up in different ways. In practice, only a very small proportion of the possible folding patterns are energetically favourable, stabilised by interactions between the ‘hooks’ protruding from the main chain. The spatial arrangement, or conformation, adopted by a chain is known as its higher-order structure and different levels of organisation can be identified. Indeed, one of the remarkable things about proteins is that any chain with the same sequence of amino acids will fold up in exactly the same way, provided the conditions are the same, although a bit of help is sometimes needed to achieve this. Thus, primary structure determines higher-order structure, which in turn, as we shall see later, determines function.

Collagen is a particularly abundant fibrous protein that accounts for 25 per cent of all body protein. It has high tensile strength and is found particularly in skin and tendons. The higher-order structure of collagen is illustrated in Figure 13. The basic structural unit is a triple helix, composed of three polymer chains (which are themselves helical) coiled round each other and held together by weak, non-covalent bonds, as shown in Figure 13(a). These units are then packed side by side in small groups of up to 200 to form larger fibrils, Figure 13(b), which are bundled together to form fibres, Figure 13(c). The fibres may themselves clump together in larger aggregates to build up body tissues. The result of all this organisation is a very strong, reasonably stiff, tough material.

One of the most powerful methods available for working out the structures of proteins is X-ray diffraction: a technique that relies on the scattering of X-ray radiation by a regular crystal lattice. It is not always easy to persuade a protein to crystallise, but as techniques improve and higher-intensity X-ray sources and more powerful analytical tools become available, more and more proteins are being characterised in this way. Computer modelling is then applied to translate a diffraction pattern to a likely molecular structure. The Protein Data Bank (PDB) is a website maintained by the Research Collaboratory for Structural Bioinformatics (RCSB), which acts as a central repository for results in this field and provides an excellent example of international cooperation between scientists. Structures for most of the proteins mentioned in this block, and interesting articles about many of them, can be found on the PDB website.

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Figure 13 A triple helix forms the basic structural unit of collagen (a), up to 200 of which are packed together to form fibrils (b), which themselves pack together to form fibres (c)

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Collagen provides us with a good example of how protein molecules can self-assemble to produce a structure well suited for a particular purpose. It can also be used to demonstrate the limitations of self-assembly. The three intertwined strands that make up the basic unit of collagen are created and assembled simultaneously within the cell. If we separate the strands – which we can do very easily by heating collagen in water above 70 °C – and then allow them to re-assemble, the triple helices will not re-form. Instead, separate strands interact with one another to form many links with other molecules and the result is a large, open network. This is exactly what happens when you make a jelly. Breaking down the natural structure of a protein is known as denaturing. The individual molecules from the denatured collagen are what we call gelatin. When a jelly sets, the chains link together to form a huge, continuous, three-dimensional network like a giant cage, which traps the water inside through hydrogen bonding to the protein molecules.

Exercise 1

Consider a short segment of protein chain, just six monomers long.

(a) What is meant by the primary structure of the chain?
(b) How many different types of monomer are available in nature?
(c) How many different sequences are possible in the six-monomer segment? Assume that all the possible monomers are available at each of the six positions.

Answer

(a) The sequence of amino acids along the chain.
(b) There are 20 naturally occurring amino acids.
(c) At each position along the chain there are 20 possibilities. So the number of possible sequences is 20⁶ = 64 000 000.

Regular helical structures like those that occur in collagen are very common in proteins, because they allow many weak bonds to form and these stabilise the structure. Figure 14 shows a schematic representation of two particularly common regular folding patterns, the α-helix and the β-sheet.

Figure 14 Schematic representation of α-helix and β-sheet structures

Long description

Keratin is another important example of a fibrous protein, with a different amino acid sequence. It can exist in different forms, based either on α-helices (in mammals) or β-sheets (in birds and reptiles). It is a major component of skin, hair, nails, hooves, horns, claws, beaks, feathers and scales, which gives a good indication both of its versatility and its strength.

More disordered network structures can also be produced, by incorporating segments in the polypeptide chain that can form cross-links to neighbouring chains. This results in a rubber-like structure with high extensibility; elastin, a key component of skin, has this structure.

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3.3 Spider silk

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The presence of regions of helical and sheet-like structures within a protein will affect its properties in different ways: a particularly striking example of this is provided by spider silk.

Imagine a polymer that forms fibres stronger, by mass, than steel and can be processed from water at ambient temperature and low pressure. As a consequence of its biological origin, it is extremely environmentally friendly and totally recyclable. It may sound like science fiction, but this is exactly what we have in spider silk. Mind you, it has taken 400 million years to develop!

Spider silk is composed of proteins. Spiders make their webs and perform other tasks using up to seven different types of silk fibre. Each silk has distinct mechanical properties, which arise directly from the primary and higher-order structure of the proteins from which they are constructed. Table 4 lists three types of silk from the golden orb web-weaving spider (Nephila clavipes) shown in Figure 15 and compares their mechanical properties to some synthetic polymer materials.

Table 4 Comparison of mechanical properties of spider silk and other polymer fibres

Material	Use	Tensile strength / MPa	Extension at breaking point / %	Energy to break / kJ kg⁻¹
Dragline silk	web frame and radii, support when climbing or falling	4000	35	100
Flagelliform silk	core fibres of adhesive spiral	1000	>200	100
Silk from the minor ampullate gland	web reinforcement	1000	5	30
Kevlar	high-strength applications	4000	5	30
Rubber	high-elasticity applications	1	600	80
Nylon 6	wide range of uses	70	200	60

(Source: adapted from Hinman H.B. et al. (2000) ‘Synthetic spider silk: a modular fiber’, Tibtech, vol. 18, no. 9, pp. 374–379.)

Not surprisingly, given its impressive properties, spider silk has been the focus of much research. Scientists have identified the major proteins that make up the different silks and have mapped their amino acid sequences. It turns out that there are just a few key repeating patterns, which in turn lead to particular secondary structures. These are summarised in Table 5: the letters G, P and A represent the amino acids glycine, proline and alanine respectively, with X as a ‘wild card’. There are two helical structures present here, known as a β-turn spiral and a 3₁₀ helix; you can think of these as acting like a loose and a tight spring. The structures also include spacers to separate the repeating units into clusters, but these aren't included in the chart.

Table 5: Structural modules found in spider silk from the golden orb spider; read across the rows to see the composition of each type


		Elastic β‑turn spiral	Crystalline β‑sheet	3₁₀ helix
Amino acid sequence		GPGXX	(GA)_n or A_n	GGX
Dragline silk	protein 1		√	√
	protein 2	√(~5 repeats)	√
Flagelliform silk		√(~50 repeats)		√
Silk from the minor ampullate gland	protein 1		√	√
	protein 2		√	√

(Source: adapted from Hinman H.B. et al. (2000) ‘Synthetic spider silk: a modular fiber’, Tibtech, vol. 18, no. 9, pp. 374–379.)

Figure 15 Golden orb spider on its web

Long description

The dragline silk, which forms the main structure of the web and also supports the spider when climbing or dangling on a thread, is exceptionally strong and reasonably elastic, making it a very tough fibre comparable to the best that modern polymer technology can offer. By contrast, the flagelliform silk of the spiral part of the web is not only sticky but very stretchy, so that it can absorb the energy from a collision with a large insect without breaking, and keep the insect trapped in the web. The silk from the minor ampullate gland is as strong as the flagelliform silk but has much lower elasticity and is used for structural reinforcement.

The correlation between the secondary structures within the proteins and the physical properties of each silk is striking, as you'll see by working through SAQ 6.

SAQ 5

Use Tables 4 and 5 to answer these questions.

(a) Which two types of silk have the highest extensibility?
(b) What structural feature do they have in common?
(c) To what would you attribute the difference in extensibility between the two?
(d) Why would you expect the β-sheet arrangement to convey strength?
(e) Suggest why a particularly regular amino acid sequence might be needed to form a β-sheet.
(f) How might you account for the high strength but low extensibility of the silk from the minor ampullate gland, by considering its structure?

Answer

(a) Flagelliform silk and dragline silk.
(b) The presence of helical structures: the elastic β-turn spiral and the 3₁₀ helix.
(c) The more elastic flagelliform silk has many more units in the elastic β-turn spiral component, i.e. it contains longer loose springs. Also there are no stiff β-sheet regions in the flagelliform silk.
(d) The β-sheet conveys strength because of the large density of hydrogen bonds between adjacent chains, which keep them fixed in position.
(e) The polypeptide chains are packed closely together in the β-sheet and the presence of different side groups would disrupt this regular arrangement.
(f) There are no elastic β-turn spiral modules in the minor ampullate silk to provide elasticity, but β-sheet regions are present to convey strength.

It isn't just the composition of spider silk that is of interest to technologists, but also the spinning process itself. The formation of secondary structural motifs such as those described result in a liquid crystalline solution, which reduces the amount of force needed by the spider to achieve high orientation. The fibres are extruded through a spinneret in the spider's abdomen, which involves a novel internal stretching process not seen in current industrial fibre processing.

The very close link between molecular-scale structure and large-scale properties seen in spider silk suggests that by carefully controlling the sequence of monomer units along a polymer chain we should be able to design materials with very closely specified properties. Indeed, polymer chemists have been doing this in a crude way for many years by designing block copolymers for specific applications. Synthetic block copolymers typically contain only two or three different monomer units, but a wide range of properties can be achieved. The potential degree of control over properties in a polypeptide chain with 20 different monomer options is much greater.

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4 Engineering with proteins

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What are the prospects for designing and making new proteins for specific purposes? The technology exists to build polypeptide chains unit by unit in a test tube, but this is time-consuming and expensive. Often a more practical approach is to find ways of working with nature to produce useful substances in a form that we can use. This might involve extracting a naturally occurring protein and chemically modifying it in some way, or using genetic engineering to produce a particular protein in controlled conditions. An example of how this has been applied to spider silk is described in Box 5 Genetic engineering: spider silk from goats?

Box 5 Genetic engineering: spider silk from goats?

Proteins are made in cells by following coded instructions carried by the genetic material, DNA. A gene is a portion of DNA that holds the instructions for a particular protein. If these instructions are changed, or added to, then different proteins can be made. Genetic engineering is the term given to a range of new research techniques that allow us to manipulate the DNA in cells.

Gene-splicing is a method for transferring selected genes from one species to another, by cutting a fragment from the DNA of one organism and joining it to a DNA molecule from another organism. Bacteria are often used as the host and can be used to produce large quantities of artificial proteins in the lab.

Spider silk can't easily be made in bulk by farming spiders, because they have an unfortunate habit of eating each other. Transferring the gene to bacteria has not proven successful either, producing silks that are impossible to spin or have low strength. Consequently, some researchers have turned to mammal cells as a possible alternative host and, in 2002, a Canadian company announced that it had succeeded in breeding goats carrying a modified version of the spider-silk gene. The protein can be extracted from the goat's milk, and spun into yarn in a process that mimics conditions in the body of the spider.

The technology is still a long way from practical production, but if development continues we could see spider silk rivalling synthetic polymers like nylon and Kevlar in specialist applications where high toughness is required, from bulletproof clothing to reinforced composites for aircraft panels.

Using naturally occurring proteins in new ways is one thing, but the ultimate goal of designing new protein-based materials or devices from scratch is much, much more difficult. I chose spider silk as an illustration of structure-property relationships within proteins because it has been widely studied and the links between the conformation of the protein chain and the mechanical properties of the material are, at least at a superficial level, relatively easy to spot. However, for most proteins, particularly those with more complex functions, such links are much harder to establish.

Designing a protein does not just involve choosing the right ingredients to achieve a desired structural element: it must also take account of possible unforeseen interactions which might lead to unwanted folded-chain conformations. This aspect is often termed negative design. The goal of negative design is to ensure that only one folded-chain conformation is formed, by ensuring that all other possible structures are energetically unfavourable. Not a trivial task!

In one simple example of protein design, scientists are working to produce improved materials for making water-based gels (hydrogels), in a similar way to that in which denatured collagen associates to form jelly. Hydrogels have many important uses, from highly absorbent dressings and water-retaining granules for agriculture, to structural devices such as contact lenses and components for microfluidic analysis systems.

The polypeptide chains designed to make the gels in my example consist of two different modules. The central portion is a long, disordered chain that cannot fold into a regular structure, but that includes hydrophilic side groups that can associate with water. At the ends are stretches of chain with a row of hydrophobic side groups. The groups from one chain can line up side by side with those from another in a leucine zipper. These form the cross-links, which are weak enough to break apart when heated. By varying the length and the precise composition of the different sections, a range of proteins with different gelation properties can be made.

However, in most cases we are a very long way indeed from being able to design a protein from scratch for a particular function. At the moment, the best we can typically hope to do is to identify the function of naturally occurring protein-based molecular devices and try to identify the key parts of the molecule and how they work. Then, if we are very lucky, we might get away with a tiny bit of tweaking in order to make the molecule fit in better with our system. Many of the current practical applications of bionanotechnology are aimed at doing just that.

SAQ 6

(a) What are the principal design requirements for a protein chain if it is to form a random, cross-linked network capable of trapping water to form a gel?
(b) Which aspects would you classify as positive design and which as negative design?
(c) What extra feature is required if the gel is required to break up on heating?

Answer

(a) Random implies no crystallisation. An irregular sequence of groups with little capacity for forming secondary bonds is required. The two protein chains must be able to link together at certain points to form a network. The chains must contain some hydrophilic groups to associate with the water molecules and keep them trapped within the network.
(b) Choosing an irregular sequence of groups that won't form secondary bonds is negative design. The linkages, and the need for some hydrophilic groups, are positive design.
(c) If the gel is required to break up on heating then the cross-links must not be permanent, but formed by weak bonds that can break when heated.

As an example of how a naturally occurring protein can be put to a new use, and modified for the purpose, let's look at green fluorescent protein (GFP), which is found in certain types of jellyfish. As its name suggests, GFP emits green light, using energy absorbed from ultraviolet light. Controversially, it has been introduced into animal cells through genetic engineering, to create fluorescent varieties of plants and animals, such as the ‘GFP bunny’ described as a ‘transgenic artwork’ by its creator, the artist Eduardo Kac. Such uses have sparked important public debate on both the safety and the morality of genetic engineering: issues that are likely to increase in significance as our knowledge and capabilities advance further.

A much less controversial application of GFPs is their use in scientific and medical research to ‘label’ an object of interest and to study its behaviour. For instance, GFP can be attached to another protein, whose movement around a cell can then be tracked simply by watching the green glow move around under a microscope. Another recent development is to modify GFPs to act as biosensors, which fluoresce in a characteristic way if a particular substance is present.

The structure of GFP is illustrated in Figure 16. This type of picture is known as a ‘ribbon representation’ as it uses ribbons to pick out key structural features. You should be able to spot a β-sheet, here folded up to produce a ‘can’. There are also short sections of a-helix, coloured red in the diagram, one of which passes down the middle of the can. In the middle of the β-can, safely protected from polar water molecules that would interfere with its action, is the light-emitting centre, shown in yellow on the diagram. This is made up from a particular sequence of three amino acids, which interact together to form a structure in which the band gaps are suitable for the absorption and emission of light.

Figure 16 Structure of green fluorescent protein: ribbon representation

Long description

We can modify this structure by making slight changes in the amino acid sequence or by adding portions to the end of the chain, while keeping enough of the original arrangement to ensure that the basic design is not compromised. How might we want to change it, to increase its usefulness? One possibility is to tweak the colour of the light emitted, by making slight adjustments to the environment of the light-emitting structure. Variations of GFP that fluoresce blue, cyan and yellow have all been made, allowing different substances to be tagged and monitored at the same time. Another common modification is to provide attachment sites for different molecules by adding flexible linkages to the ends of the polypeptide chain, so that the range of substances that can be labelled is increased. In another example, a blue fluorescent protein has been developed to detect the presence of zinc ions; zinc ions interact with the light-emitting centre in a way that increases the level of fluorescence, so that the level of zinc present can be measured from the brightness of the signal.

This is just one example of how we can learn from nature by taking a naturally occurring molecular machine and extending its function to suit our own goals. As we work our way through the cell we will see many more biological devices and learn further principles for working at the nanoscale, which we may be able to adapt and apply to new situations. One day we will surely know enough to design novel nanomachines of our own.

SAQ 7

(a) Describe aspects of the primary and higher-order structure of the GFP protein that make it particularly suited for its purpose as a light emitter.
(b) Which parts of the structure would you expect to be most open to modification, without compromising the function of the molecule?

Answer

(a) The key feature of the primary structure is the sequence of three amino acids that forms the light-emitting centre. The key features of the higher-order structure are the β-can, which protects the light-emitting centre from the polar water molecules that surround the structure and would interfere with its operation, and the α-helix, which positions the light-emitting centre in the right place in the centre of the can (both of these would, of course, require a suitable primary sequence of amino acids to form, but different variations are possible).
(b) The loose loops at the top and bottom of the structure, and the chain ends, would be the obvious places to attempt to modify the structure, as their precise shape is likely to be less crucial to the functioning of the device.

Exercise 2

Suggest an engineering application that could benefit from the manufacture of protein-based structural materials.

Answer

The example I thought of was tissue engineering, where biological materials are being developed to provide substitutes for living tissue – for instance, to replace skin or to repair damaged blood vessels. An ultimate aim is to find ways to prompt the body to grow its own replacement parts.

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Acknowledgements

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Grateful acknowledgement is made to the following sources:

Figures

Figure 7 (a): PDB ID 1BKV Kramer, R. Z., Bella, J., Mayville, P., Brodsky, B. and Berman, H. M. (1990) ‘Sequence dependent conformational variations of collagen triple-helical structure’, Natural Structural Biology, vol. 6, pp. 454–57

Figure 7(b): PDB ID 1ATN Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. and Holmes, K. C. (1990) ‘Atomic structure of the actin:DNase I complex’, Nature, vol. 347, pp. 37–44

Figure 7(c): PDB ID 5PEP Cooper, J. B., Khan, G., Taylor, G., Tickle, I. J. and Blundell, T. L. (1990) ‘X-ray analyses of aspartic proteinases. II. Three-dimensional structure of the hexagonal crystal form of porcine pepsin at 2.3 A resolution’, Journal of Molecular Biology, vol. 214, pp. 199–222

Figure 7(d): PDB ID 2OHX Al-Karadaghi, S., Cedergren-Zeppezauer, E. S., Petratos, K., Hovmoeller, S., Terry, H., Dauter, Z. and Wilson, K. S. ‘Refined Crystal Structure of Liver Alcohol Dehydrogenase-Nadh Complex at 1.8 Angstroms Resolution’ [to be published]

Figure 7(e): PDB ID 2POR Weiss, M. S. and Schulz, G. E. (1992) ‘Structure of porin refined at 1.8 A resolution’, Journal of Molecular Biology, vol. 227, pp. 493–509

Figure 7(f): PDB ID 1BL8 Doyle, D. A, Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T. and MacKinnon, R. (1998) ‘The structure of the potassium channel: molecular basis of K⁺ conduction and selectivity’, Science, vol. 280, pp. 69–77

Figure 7(g): PDB ID 1EUL Toyoshima, C, Nakasako, M., Nomura, H. and Ogawa, H. (2000) ‘Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution’, Nature, vol. 405, pp. 647–55

Figure 7(h): PDB ID 2HIU Hua, Q. X, Gozani, S. N., Chance, R. E, Hoffmann, J. A., Frank, B. H. and Weiss, M. A. (1995) ‘Structure of a protein in a kinetic trap’, Natural Structural Biology, vol. 2, pp. 129–38

Figure 7(i): PDB ID 1GG2 Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G. and Sprang, S. R. (1995) ‘The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2’, Cell, vol. 83, pp. 1047–58

Figure 7(j): PDB ID 1FXK Siegert, R., Leroux, M. R., Scheufler, C, Hartl, F. U. and Moarefi, I. (2000) ‘Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins’, Cell, vol. 103, pp. 621–32

Figure 7(k): PDB ID 1RVA Kostrewa, D. and Winkler, F. K. (1995) ‘Mg²⁺ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution’, Biochemistry, vol. 34, pp. 683–96

Figure 7(1): PDB ID 1TAU Eom, S. H., Wang, J. and Steitz, T. A. (1996) ‘Structure of Taq polymerase with DNA at the polymerase active site’, Nature, vol. 382, pp. 278–81

Figure 7(m): PDB ID 1B7T Houdusse, A., Kalabokis, V. N., Himmel, D., Szent-Gyorgyi, A. G. and Cohen, C. (1999) ‘Atomic structure of scallop myosin subfragment SI complexed with MgADP: a novel conformation of the myosin head’, Cell (Cambridge MA), vol. 97, pp. 459–70

Figure 15 From Isaac Leung's page, The University of British Columbia.

Figure 16 Structure of Green Florescent Protein. Taken from the Department of Biochemistry and Cell Biology, Rice University, Houston.

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Module team

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T356 course team

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