1. Introduction
There are relatively few carbohydrates; there are also limitless numbers of proteins. They are specific to each species. Glucose is glucose in whatever organism it occurs, but proteins vary from one species to another. Indeed, it is proteins rather than the fats or carbohydrates that determine the characteristics of a species.
Proteins are organic compounds of large molecular mass (rmm ~ 20,000 to 10,000,000 Da). Comprise over 50% of total dry mass of the cell. In addition to carbon, hydrogen and oxygen, they also contain nitrogen, often sulphur and phosphate (sometimes). Proteins are the most structurally sophisticated molecules known. Consistent with their diverse function, they vary extensively in structure, each type of protein having a unique 3D shape or conformation.
Proteins are all polymers constructed form the same set of 20 amino acids. Polymers of a linear sequence of amino acids are caller polypeptide chains. A protein is functional and may consist of 1 or more polypeptide chains folded and coiled into a specific conformation. There are 2 key aspects of proteins that are important for the understanding of its function. They are, the nature of amino acids based on their side chain groups and the shape of the protein macromolecule called its conformation.
2. Classification of Proteins
It can be based on its function. For example, structural, enzymatic function, hormones, transport, protective, contractile, storage, toxins etc.
Based on composition, a protein can be divided into 2 general classes. Simple proteins which yield only amino acids on complete hydrolysis. And conjugated proteins which on degradation, yield in addition to amino acids, an organic or inorganic non-peptide group termed as a prosthetic group.
Finally, they can be grouped based on their shapes. Each protein has a unique 3D shape and the protein’s function depend on this shape.
3. Amino Acids
The most important role of amino acids is as the monomers which are polymerized to make proteins. Amino acids can also be used as intermediates for synthesis of other molecules and they can be used as a source of energy if there are no other sources available.
Plants are able to make all the amino acids they require from simpler substances, such as carbohydrates and the nitrates that they absorb. However, animals are unable to synthesize all that they need and therefore must obtain aome ‘ready-made’ amino acids directly from their diet. These are termed as essential amino acids.
There are 20 different fundamental amino acids. In addition to these, are derivatives of these amino acids known as rare amino acids, for example, hydroxyproline and hydroxylysine. There is no triplet code for rare amino acids and they are derived from the modification of their parent amino acids after incorporation into the polypeptide chain (post translational modification).
The chemical compositions of amino acids are carbon, hydrogen, oxygen, nitrogen and sulphur. Amino acids consist of a chiral carbon atom bonded to 4 different groups. These are the basic amino group, an acidic carboxyl group, a hydrogen atom and a variable R group (the side chain).
With exception to glycine, all amino acids have an asymmetrical carbon atom and therefore exhibit optical isomerism.
3.1 Properties of Amino Acids
Amino acids are colourless and crystalline solids with relatively high melting points.
They are able to form zwitterions. They are generally insoluble in organic solvents but soluble in water where they form ions. These ions are formed by the following process. The loss of a hydrogen ion from the carboxyl group making it negatively charged. This hydrogen ion associates with the amino group making it positively charged. This ions are therefore dipolar and are called zwitterions.
Since they
exist as zwitterions in aqueous medium, amino acids therefore have both acidic
and basic properties i.e. they are amphoteric. Being amphoteric means that
amino acids act as buffer solutions which resists the alteration of pH of the
solution when small amounts of acid or alkali are added to it. In an acidic
medium, the carboxyl group accepts a proton so the overall molecule becomes
positively charged. In an alkaline medium, the amino group loses a proton (to
combine and neutralize the
Amino acids are distinguished from one another by their R groups. These groups have important physical and chemical properties that determine the properties of the protein they make up.
3.2 Classification of Amino Acids
3.2.1 Based on Human Nutrition
Essential amino acids cannot be synthesized from simpler substances and must be obtained from diet. Continuous deprivation of even one or two essential amino acids can be disastrous. Non-essential amino acids can be made form simpler substances by humans.
3.2.2 Based on Functional Characteristics of the R Group
The unique characteristic of a particular amino acid is determined by the physical and chemical properties of its R group. Polar amino acids are amino acids with polar side groups (ions and polar uncharged molecules) are hydrophilic. Acidic amino acids are those with negatively charged side chains; owing to the presence of carboxyl group in the side chain (hydrophilic) Basic amino acids have positively charged amino groups in their side chain (hydrophilic). Non-polar amino acids are those with non-polar side groups which are hydrophobic and underactive.
3.2.3 Based on Chemical composition of R groups
These are grouped according to aliphatic, hydroxyl, sulphur, aromatic, acidic, basic and imino.
4. Peptide Bonds
This covalent bond is formed between the amino group of one amino acid and the carboxyl group of an adjacent amino acid. In the formation of the peptide bond, a water molecule is eliminated. This is a condensation reaction and the linkage formed is a covalent carbon-nitrogen bond called the peptide bond. Further combinations of this type extend the length of the chain to form a polypeptide.
A polypeptide chain has 2 terminuses. The N-terminal has the free amino group while the C-terminal has the free carboxyl group.
Proteins with different functions usually have very different amino acid sequences. Proteins with similar functions have varying degrees of similar sequences. Sequence comparison determines which part of the polypeptide is crucial for their function. Same proteins performing the same function occuring indifferent species have extensive similarities in sequences.
As a crude rule, a protein is functional, while polypeptides are often components of a protein. A protein is a functional unit composed of 1 or more linear unbranched polypeptide chains. All molecules of a given type of protein are identical in amino acid composition, sequence and length of a polypeptide. Very importantly, a protein’s conformation is 3D and the protein’s function is dependent on its unique shape.
5. Levels of Protein Structure
A protein has information in the form of its amino acid sequence and it is this sequence that determines the 3D conformation that the polypeptide will assume to become functional. It is this specific conformation that determines how the protein works.
This shape is important in the functioning of proteins. This 3D conformation of a polypeptide is maintained primarily by 4 types of bonding which occurs between various amino acids in the chain.
The sequence in which the amino acids are linked together determines the primary structure of a protein; the way in which the resulting chain bends of folds is the secondary structure. Further bending and folding create the tertiary structure and in some complex proteins, the physical relation of separate chains makes up the quaternary structure.
The folding occurs because the side chain groups of the different amino acids react in a variety of ways with each other and with the environment.
5.1 Primary Structure
The primary structure of a polypeptide is its unique number and linear sequence of amino acids. This precise primary structure is determined not by random linking of amino acids but specified by nucleotide sequences in genes (coded in the form of triplet codons on DNA).
The primary structure contains information for the protein’s folding into a specific 3D shape. Sequence of amino acids determines where the alpha helixes or beta pleated sheets can occur, where disulphide bonds are located and so on.
In sickle cell anemia, a slight change in one amino acid can affect a protein’s conformation and ability to function. In this case, a polar glutamic acid is substituted by a non-polar valine in position 6 of one of the polypeptide chain.
5.2 Secondary Structure
Most proteins have segments of their polypeptide chain repeatedly coiled or folded into a specific shape that contribute to the protein’s overall conformation. The secondary structure is the regular arrangement/folding of the regions of the polypeptide chain with common repeating structural patterns as a result of hydrogen bonds formed at regular intervals at polypeptide backbones.
Hydrogen bonds are bonds that are individually weak but collectively serve to stabilize the structure. Examples of secondary structure are alpha helix and beta pleated sheets. These are structures that are more rigid than the extended unfolded polypeptide. Both these types of conformation may occur within the same polypeptide.
5.2.1 Alpha Helix
In many proteins, parts of the polypeptide chains take the form of a alpha helix which is the form of an extended spiral spring. It is maintained by numerous hydrogen bonds which are formed between C=O groups (on the nth amino acid) and the NH group (on the nth + 4 residue) between every 4th peptide bond. The hydrogen of the peptide amino group forms a hydrogen bond with the oxygen of the peptide carbonyl group of the 4th peptide bond. Intramolecular bonds are parallel to the axis of the helix, so maximally stable. The alpha helix makes one complete turn for every 3.6 amino acids. Each peptide bond participates in hydrogen bonding conferring max stability.
Hydrophobic R groups of the amino acid residues points outside the helix, perpendicular to the main axis. Since all of the main chain peptide N and carbonyl O residues are hydrogen bonded this greatly reduces the hydrophilic nature of the helical region. This structure is hydrophobic, flexible and elastic.
Proline and hydroxyproline inserts a kink and disrupts the formation of a smooth helical structure. Polyproline sequences are unable to assume any secondary structures as the kink that results presents conformational constraints. Amino acids with bulky side groups, if present in large numbers, can also interfere with the formation of the alpha helix.
A protein, which is entirely alpha helix, and hence fibrous is alpha keratin. It is the structural protein of hair, wool, nails, claws and horns. It is composed of 3 or 7 such helices coiling round each other to form 3-stranded or 7-stranded ropes held together by disulphide bonds. This produces very strong fibres. Alpha keratin is tough. The hardness and stretchability vary with the degree of cross-linking by disulphide bonds between neighbouring polypeptide chains. It is also insoluble in water. At pH 7.0 at room temperature, it is insoluble because it is rich in amino acids with hydrophobic R groups that are exposed to the aqueous environment outside of the fibre.
Springiness of hair and wool fibres is a consequence of the tendency of the coiled coil alpha helix to untwist when stretched and to recover its original conformation when the external force is relaxed. However, hard alpha keratins are less pliable because of the high number of disulphide bridges that resist any forces trying to deform them.
5.2.2. Beta Pleated Sheet
This is the case in which a single polypeptide chain folds back and forth, or when 2 regions of the chain lie either parallel or anti-parallel to each other. ‘Side-by-side’ polypeptides form sheets.
This structure is stabilized by large numbers of hydrogen bonds. These bonds are formed between the C=O and NH groups of one chain and the C=O and NH groups of adjacent chains/regions. Hydrogen bonding in sheet structures can occur among 2 or more segments of the same chain (intrachain sheet) or among 2 or more segments of different chains (interchain sheets) Bulky amino acids interfere with the formation of such structures, thus amino acids have compact R groups.
Silk fibroin, the protein used by silk worms when spinning their cocoon threads, is entirely in this form. This structure involves a number of adjacent chains; they are arranged in anti-parallel fashion. The sheet has high tensile strength and cannot be stretched. Silk fibroin is flexible but inelastic, very supple, has high tensile strength and antiparallel.
5.3 Tertiary Structure
Tertiary structure refers to the precise overall 3D shape of a protein. It is concerned with the bending, twisting and folding of the secondary structure to give the 3D shape. This may involve stearic/spatial relation of residues that are far apart on the polypeptide chain as well as those that are adjacent.
A protein’s tertiary structure and thus the protein’s stability is maintained by mainly 4 types of bonding, which occurs between various amino acids in the chain. This includes the strong covalent disulphide bond, the ionic bond and the 2 weaker interactions of hydrogen bonds and hydrophobic interactions.
The secondary structure describes the interactions between the different parts of the polypeptide backbone, whilst the tertiary structure describes the interactions between the R groups of the amino acids.
Disulphide bonds are formed by the oxidation of 2 sulphydral groups (SH) of adjacent amino acids in the same or in different polypeptide chains. These bonds can be cleaved by reducing agents. Hair so treated can be curled and set in ‘permanent wave’ by application of oxidizing chemicals which re-establishes the disulphide bonds in the ‘new curl’ conformation.
In acidic amino acids, the COOH groups on the side chain may ionize to give COO- groups, and in basic amino acids, NH3+ groups are found on the side chains. These oppositely charged groups may form ionic bonds which help to give the polypeptide its particular shape. These ionic bonds are weak and may be broken by the addition of other ions or the alterations in the pH of the medium around the polypeptide.
Hydrogen bonds occur between certain hydrogen atoms and oxygen atoms within the polypeptide chain. The hydrogen atoms have a small positive charge and the oxygen atoms have a small negative charge. The 2 charged atoms are attracted together to form a weak hydrogen bond. Each bond is relatively weak but the large number of bonds makes a considerable fold in the shape and stability of a polypeptide molecule.
Hydrophobic interactions are interactions between non-polar R groups. These causes the proteins to fold as hydrophobic side groups have the tendency to associate with one another and be shielded from water. This is most important when the protein folds so as to shield the hydrophobic side groups from the aqueous environment at the same time exposing the hydrophilic side chains.
Cysteine is involved in disulphide bonds while proline inserts a kink and disrupts alpha helices in the polypeptide chain.
5.3.1 Myoglobin
All globular proteins exhibit the following 3 common features. They show compact folding. There is little or no room for water molecules in the interior. Nearly all hydrophilic R groups are on the surface; hence it is soluble in water. Half or more of the hydrophobic R groups are located internally and are out of contact with water. The stabilization of tertiary structure in globular proteins is achieved through non-covalent interactions.
Myoglobin is a haem protein that functions both as a reservoir or a store for oxygen and as an oxygen carrier, releasing oxygen readily when the cells require it. It is present mainly in the muscles and heart which require large amounts of oxygen because of their need of large amounts of energy during contraction.
Myoglobin is a single polypeptide of 153 aa. No quaternary structure. It consists of 8 segments of alpha helices and bent to form a compact globular structure that is soluble in water. The same 4 interactions that stabilize the tertiary conformation apply to myoglobin. The interior of the molecule is composed almost entirely of non-polar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions. Charged and polar amino acids are located on the surface where they can form hydrogen bonds with water. Myoglobin is a conjugated protein with a haem prosthetic group in the hydrophobic cleft.
Haem is a complex of porphyrin rings with a ferrous iron at its centre. The haem is held in place within the folded polypeptide by weak bonds between the R groups
5.4 Quaternary Structure
Many highly complex proteins consists of 2 or more polypeptide chains held together by hydrophobic interactions, hydrogen bonds, disulphide bonds and ionic bonds into one functional protein. Quaternary structure is the overall protein structure that results from the association of polypeptide subunits to form a biologically functional protein.
5.4.1 Haemoglobin
Haemoglobin is found exclusively in red blood cells. Its main function is to transport oxygen from the lungs to the capillaries of the tissues.
It is a tetrameric protein that consists of 4 polypeptide chains, namely 2 alpha chains (141 aa) and 2 beta chains (146 aa). They are held together by non–covalent interactions.
Each subunit has stretches of alpha helical structure and a haem-binding pocket similar to that of myoglobin. Each subunit is a conjugated polypeptide that is associated with a prosthetic group. The haem groups are responsible for its red colour and are the sites of oxygen transport.
The tetramer can be said as being composed of 2 identical dimers. (Alpha Beta 1) and (Alpha Beta 2). The 2 polypeptide chains in each dimer are held together by interchain hydrophobic interactions however, ionic and hydrogen bonds also occur.
The 2 dimers are held together by weak polar interactions resulting in the ability of the 2 dimers to move with respect to each other. This allows the 2 dimers to occupy different relative position in deoxyhaemoglobin compared to oxyhaemoglobin.
The iron atom of the haem group combines with a molecule of oxygen without the oxidation of iron (II). The oxygen molecules fits into the pockets called binding sites. Up to 4 oxygen molecules can be carried by each heamoglobin molecule and the oxygen binds reversibly.
The binding of 1 oxygen molecule to one of the subunits induces the remaining subunits to change their shape slightly so their affinity increases to take more oxygen easily. Thus the hesitant loading of the first oxygen molecule results in the rapid loading of 3 more, When one subunit unloads its oxygen, the other 3 quickly follow the lead as a conformational change lowers their affinity for oxygen. This mechanism is known as coorperativity.
Haemoglobin is an allosteric protein. Having 4 interacting subunits gives it certain properties that are essential to its role as an oxygen carrier. Haemoglobin must bind oxygen at one place and give up oxygen at another. This process is more efficient if haemoglobin travels fully charged and dumps all its oxygen at a single location.
5.4.2 Collagen
Collagen is a fibrous protein and is a major protein of vertebrate and is found extensively in connective tissues, bones, cartilage and dermis. It consists of 3 polypeptides called alpha chains wound around each other in a triple helix, forming a rope-like structure.
Each individual alpha chain has a primary structure of 1000 aa residues and is largely a repeat of a tripeptide sequence Gly-X-Y, where X is often a proline and Y is a hydroxylysine or a hydroxyproline (important in stabilizing the triple helix structure). One third of the aa residues are the smallest aa and is found in every 3rd position, which fits into the restricted space where the 3 chains of the helix come together.
3 collagen polypeptide chains are then wound together to form a right-handed triple helix called tropocollagen. Cross bridges linking the 3 polypeptide chains provide additional structural support. Due to this organization, collagen has great tensile strength and cannot be stretched, which is essential for its functioning.
Tropocollagen molecules assemble to form collagen fibrils which further assemble to form collagen fibres. Each collagen fibre is about 3000 angstroms in length with an approximate molecular weight of 300 kDa.
6. Denaturation and Renaturation of Proteins
Denaturation of proteins is the loss/alteration of the specific 3D structure of a protein molecule. This change may be temporary or permanent but the aa sequence of the protein remains unaffected. Once denaturation occurs, the molecule unfolds and can no longer perform its normal biological function.
Protein denaturation can be caused by several of the following factors
Most proteins become denatured if they are transferred from an aqueous environment to an organic solvent. The protein turns inside out, the hydrophobic regions change places with the hydrophilic regions.
Other agents of denaturation include chemicals that disrupt the hydrogen bonds, ionic bonds and disulphide bonds that maintain the protein’s shape.
Denaturation can also result from excessive heat which agitates the polypeptide chain enough to overcome the weak interactions that stabilizes conformation. This breaks every bond except the disulphide bonds.
Drastic alteration of pH will also disrupt ionic bonds involved in the maintenance of the protein’s conformation.
Sometimes a protein will spontaneously refold into its original structure after denaturation, provided conditions are favourable. This is known as renaturation. This restoration of both conformation and biological activity provides evidence that the secondary and tertiary and quaternary structures are pre determined by primary structure.
