Primary structure refers to the amino acid sequence of the protein's polypeptide chain or chains, if the protein consists

of more than one polypeptide. Each amino acid residue is linked to the next via a peptide bond. The residues at the two ends of the polypeptide participate in just one peptide bond -- The amino acid residues with a free amino group and free carboxylate group are called the Nterminus and C-terminus respectively. Variations in the length and amino acid sequence of polypeptides contribute to the diversity in the shape and biological functions of proteins. Because each amino acid residue has unique chemical properties, its presence at a particular position on a protein influences the properties of that protein. With 20 different choices available for each amino acid residue in a polypeptide, a huge number of protein molecules are possible. For a protein of n residues, there are about 20^n possible sequences. In general, polypeptides usually contain between 100 and 1000 residues. The size range in which most polypeptides fall in is probably because a polypeptide chain cannot be too small and 40 residues appear to be near the minimum for a polypeptide chain to fold into a discrete and stable shape that allows it to carry out a particular function. Also, too large a chain will result in a longer corresponding mRNA and gene, leading to a greater likelihood of introducing errors during transcription and translation. The sequence of amino acids in a polypeptide, i.e. its primary structure, is encoded by the sequence of nucleotides in the corresponding gene. A specific sequence of nucleotides in DNA is transcribed to mRNA, which is then read by the ribosome in a process known as translation. The polypeptide chain is then synthesized by the stepwise polymerization of amino acids. Homologous proteins are proteins that share a significant degree of sequence similarity, or perform the same function in different organisms. We can draw conclusions about evolutionary relationships by comparing the amino acid sequences of homologous proteins. For example, cytochrome c of the electron transport chain is a protein found in nearly all eukaryotes. Amino acid sequencing of cytochrome c across species has revealed that the number of amino acid differences between two cytochrome c sequences from two different species is proportional to the phylogenetic differences between the species from which they are derived. This implies that a phylogenetic tree can be constructed by analysing the sequences to find out the minimum number of mutational changes connecting the branches. This can be used to infer ancestry, and hence evolution trees can be constructed solely on the basis of amino acid differences occurring in the primary sequence of one selected protein. In this case, high degrees of sequence similarity would suggest common ancestry. Another example would be oxygen-binding heme proteins in humans. Human myoglobin and human alpha-globin chain show 38 common amino acid identities, whereas human alpha-globin and human beta-globin have 64 residues in common. The relatedness suggests an evolutionary sequence of events in which chance mutations led to amino acid substitutions and divergence in primary structure. The ability to bind oxygen via a heme prosthetic group is retained by all three

polypeptides. In this case, gene duplication has given rise to a set of proteins in which the biological function has been highly conserved. Serine proteases (which are so called because serine residues play a central role in the proteins catalytic activity) show significant sequence homology. This suggests that they arose via duplication of an ancestral serine protease gene, although their substrate preferences are now quite different. Mutations in proteins can be caused by missense mutations, nonsense mutations or frame shift mutations. Replacement of DNA bases can be silent mutations, if the change occurs in an intron or if the new codon codes for the same amino acid as the original one. Missense mutations, however, result in a changed amino acid residue and a mutated protein. Nonsense mutations cause the protein to be terminated prematurely and become nonfunctional. It is also possible that a stop codon mutates into a codon for an amino acid residue, elongating the chain. Additions or deletions of whole codons results in the deletion or addition of a corresponding number of amino acid residues. However, a deletion or insertion of numbers of bases other than three shifts the reading frame during translation, which can result in more profound effects, where the amino acid sequence in the C-terminal direction changes from the point of mutation. Besides mutations, gene duplication can also increase the diversity of protein functions. When a DNA sequence containing a particular gene is copied twice, there can be selective pressures to maintain two or more copies of the same gene, especially if the protein is one that is needed in large amounts. Alternatively, the two copies can evolve independently, resulting in proteins with different functions. Multidomain proteins can also result when two or more initially independent genes fuse. Exons can be inserted into an intron region which results in exon genes being transcribed together, with a hybrid intron between them. Introns provide a domain for exon reshuffling. The splicing of introns result in more exons, which leads to more possible combinations for more proteins. Consequences of missense mutations can be illustrated through human hemoglobin variants, one of which would be sickle-cell hemoglobin. In sickle-cell anaemia, a glutamic acid residue has been replaced by a valine. The hydrophobic valine can fit into a pocket of a beta-chain in another hemoglobin molecule. In this way, hemoglobin molecules fit together into long rodlike structures that can block capillaries and cause inflammation. Thalassemia also illustrates the consequences of mutuations. The condition of thalassemia can arise in several ways: 1. One or more of the genes coding for hemoglobin chains has been deleted. 2. All genes may be present, but one or more may have undergone a nonsense mutation to produce a shortened chain, or a frameshift mutation 3. All genes may be present, but a mutation may have occurred outside the coding regions, leading to either a block in transcription or improper processing of the pre-mRNA to produce mRNA.

In case 1 or 2, no functional protein will be produced. In case 3 there may be limited transcription and translation of the correct polypeptide sequence. A protein s secondary structure is the localized, repetitive coiling and folding of the polypeptide chain. It is the local spatial arrangement of the backbone atoms. These coils and folds are the result of hydrogen bonds between backbone amide hydrogen and carbonyl oxygen. There are two main secondary structures for proteins. The first is the alpha-helix. It is an extended spiral spring that is stabilized by the hydrogen bonds between the peptide linkages in the polypeptide chain. The alpha-helix is right-handed, that is it turns in the direction that the fingers of a right hand curl when the thumb points in the direction that the helix rises. The oxygen atom in a peptide linkage is bonded to the hydrogen atom of the peptide linkage 4 amino acids away. Thus, there are 3.6 amino acids residues in one complete turn of the helix. In the helix, amino acid side chains project outward and downward from the helix, thereby avoiding steric interference with the polypeptide backbone and with each other. The core of the helix is tightly packed, that is, its atoms are in Van der Waals contact. The other main type of secondary structure is the beta-pleated sheets. It consists of two or more regions of the polypeptide chain lying parallel to each other, forming flat, zigzag sheets. The sheets are held together by intra-molecular hydrogen bonds (between peptide linkages) between the sheets. These sheets can run in the same direction (parallel) or in opposite directions (anti-parallel). Beta sheets contain 2 to >12 polypeptide strands, with an average of 6 strands. Each strand may contain up to 15 residues, the average being 6 residues. Parallel Beta sheets containing less than 5 strands are rare. This suggests that parallel beta sheets are less stable than antiparallel Beta sheets, possibly because the hydrogen bonds of parallel sheets are distorted compared to those of the antiparallel sheets. Beta sheets containing mixtures of parallel and antiparallel strands frequently occur. Other less common secondary structures are the 3.10 helix and the pi helix. These structures are similar to alpha-helix, the difference being how tightly twisted they are. For example, the chain can be tightly twisted such that each residue forms hydrogen bonds with the third successive residue, rather than the fourth. Such a helix has exactly three residues per turn. It is called a 3.10 helix because each hydrogen bond creates a 10-membered ring and there are 3 residues per turn. Untwisting the helix so as to align groups on the fifth following residue will create a pi helix. Segments with regular secondary structures such as alpha-helices or beta-sheets are typically joined by stretches of polypeptide that abruptly change direction. This stretches are called tight turns or reverse turns and almost always occur at protein surfaces. Most tight turns involve 4 amino acid residues but there can be between 2 to 6 amino acid residues in one tight turn.

Tight turns can be classified according to the number of amino acid residues they contain, categorized as delta-turn, gamma-turn, beta-turn, alpha-turn, and pi-turn, which are formed by two, three, four, five and six-amino-acid residues respectively. A strand of polypeptide in a beta-sheet may contain an "extra" residue that is not hydrogen-bonded to a neighboring strand, producing a localized disruption of the beat sheets which is known as known as a beta bulge. This allows the protein to accommodate an extra amino acid residue while still maintaining the overall architecture of the protein. A protein s tertiary structure is the folding and bending of a single protein molecule into a precise, compact, 3-dimensional shape that is unique to the protein. This shape is held in place by a few different types of interactions between the side chains(R groups) of the various amino acids. They are hydrophobic interactions, ionic bonds, disulfide bridges and hydrogen bonding. Protein structures are governed primarily by hydrophobic effects and, to a lesser extent, by interactions between polar residues and other types of bonds. [FYI: Perhaps surprisingly, hydrogen bonds, which are central features of protein structures, make only minor contributions to protein stability. This is because hydrogen-bonding groups in an unfolded protein form energetically equivalent hydrogen bonds with water molecules. Nevertheless, hydrogen bonds are important determinants of native protein structures, because if a protein folded in a way that prevented a hydrogen bond from forming, the stabilizing energy of that hydrogen bond would be lost. Hydrogen bonding therefore fine-tunes tertiary structure by "selecting" the unique native structure of a protein from among a relatively small number of hydrophobically stabilized conformations.] The hydrophobic effect, which causes nonpolar substances to minimize their contacts with water, is the major determinant of native protein structure. As a polypeptide folds into its functional shape, amino acids with hydrophobic side chains usually end up in clusters at the core of the protein, out of contact with water. The hydrophilic, polar groups face the water outside. A hydrophobic interaction is thus caused by the exclusion of non-polar substances by water. (FYI: van der Waals forces can help hold the non-polar amino acid side chains together, but this is a weak interaction) The combined hydrophobic and hydrophilic tendencies of individual amino acid residues in proteins can be expressed as hydropathies. The greater a side chains' hydropathy, the more likely it is to occupy the interior of a protein and vice versa. Hydropathies are good predictors of which portions of a polypeptide chain are inside a protein, out of contact with the aqueous solvent, and which portions are outside. Site-directed mutagenesis experiments in which individual interior residues have been replaced by a number of others suggest that the factors that affect stability are, in order, the hydrophobicity of the substituted residue, its steric compatibility, and last, the volume of its side chain.

Another interaction is the ionic bonds formed between the ionised acidic COO- side chains and the ionised basic NH3+ side chains. COO- groups can be found on the R groups of acidic amino acids and NH3+ groups found on the R groups of basic amino acids. These two groups can also be found at the ends of the polypeptide chain. Disulfide bridges help further reinforce the structure of a protein. They are formed by the oxidation of the sulfhydryl groups (SH) on the side chains of two cysteine monomers, and can be within or between polypeptide chains. Essentially, the sulfur of one cysteine bonds to the sulfur of the second. Some polypeptides whose Cys residues have been derivatized to prevent disulfide bond formation can still assume their fully active conformations, suggesting that disulfide bonds are not essential stabilizing forces. They may, however, be important for "locking in" a particular backbone folding pattern as the protein proceeds from its fully extended state to its mature form. Disulfide bonds are rare in intracellular proteins because the cytoplasm is a reducing environment. Most disulfide bonds occur in proteins that are secreted from the cell into the more oxidising extracellular environment. The relatively hostile extracellular world (e.g. uncontrolled temperature and pH) apparently require the additional structural constraints conferred by disulfide bonds. The quaternary structure of a protein refers to the association of two or more polypeptide chains into one functional complex protein molecule. Each polypeptide is referred to as a subunit and the subunits are held together by various bonds, including ionic bonds, hydrogen bonds, disulfide bonds and hydrophobic interactions. A common example of a protein that has a quaternary structure is haemoglobin. The molecules of hemoglobin that carry oxygen in human blood are tetramers made from a pair of each of two kinds of polypeptide chains. These chains are designated alpha and beta. Each of these chains has a tertiary structure much like that of myoglobin, which is made from only one polypeptide chain. However, they differ from myoglobin in that they are built to associate with each other in particular ways. This will be covered later on in the presentation. There are several structural and functional advantages to quaternary association. Firstly, for stability. One general benefit of subunit association is a favourable reduction of the proteins surface-to-volume ratio. The surface-to-volume ratio becomes smaller as the radius of any particle or object becomes larger. Because interactions within the protein usually tend to stabilise the protein energetically and because the interaction of the protein surface with water is often energetically unfavourable, decreased surface-to-volume ratio usually results in more stable proteins. Subunit association may also serve to shield hydrophobic residues from water. Subunits that reorganise themselves or other subunits avoid any errors arising in genetic translation by binding mutant forms of the subunits less tightly. Secondly, for genetic economy and efficiency. Oligomeric association of protein monomers is genetically economical for an organism. This is because less DNA is required to code for a monomer that can then assemble into a larger protein of the same molecular mass. Another way to look at this is to see that all of the information that determines oligomer assembly and interaction between

subunits is contained in the genetic material needed to code for the monomer. For example, HIV protease, an enzyme that is a dimer of identical subunits, performs a catalytic function similar to homologous cellular enzymes that are single polypeptide chains of twice the molecular mass. Thirdly, many enzymes derive at least some of their catalytic power from oligomeric associations of monomer subunits. For example, the formation of the oligomer may bring all the necessary catalytic groups together to form an active enzyme. The dissociated monomers are thus inactive. Oligomeric enzymes may also carry out different but related reactions on different subunits. Proteins can be classified into two groups based on their structures: Fibrous proteins are made up of long polypeptide chains forming long fibres or sheets. Length of polypeptide and sequence of amino acids may vary slightly between two samples of the same protein. These proteins are usually insoluble in water and provide structural support for the cell. On the other hand, globular proteins are made up of polypeptide chains folded into spherical shape. The length of polypeptide and sequence of amino acids are always identical between two samples of the same protein. These proteins are usually soluble in water and have various functions, such as for transport or as enzymes. A common example of a fibrous protein is collagen. Collagen is the most abundant fibrous protein in the human body, and is found in connective tissue in the tendons, bone, skin and teeth. Its structure consists of three alpha-helical polypeptide chains wound around each other, called a tropocollagen. Hydrogen bonds are formed between NH groups on glycine residues in one chain and CO groups of proline residues in an adjacent chain. Each chain contains about 1000 amino acids and almost every third is a glycine - the smallest amino acid. Glycine gives tropocollagen a less bulky structure, thus making it easier to form a tight coil. Each three-stranded tropocollagen then crosslinks with neighbouring tropocollagen running parallel to it, forming fibrils. Parallel fibrils then form fibres. This unique triple helix structure and formation provides collagen with great tensile strength. A good example of a globular protein is haemoglobin. To continue from just now, haemoglobin consists of four polypeptide subunits; 2 alpha chains and 2 beta chains. The alpha chains bind to the beta chains, and vice-versa, resulting in a tetrahedral molecule with the four subunits in close contact, except for a hole in the center that is freely accessible to water. Unlike myoglobin, the haemoglobin subunits have many additional hydrophobic residues exposed. These hydrophobic regions can only be covered by bringing the subunits together. They also have different polar side chains at locations where they form hydrogen bonds between subunits. It is the additional surface charge and the lack of hydrogen bonding that keep the myoglobin molecules separated.

Each subunit has a non-polypeptide component, called heme, with an iron(II) ion that binds oxygen. This means that one haemoglobin molecule has four heme groups and can thus carry up to four oxygen molecules. In its final arrangement, the hydrophobic amino acid residues of the haemoglobin are buried in the interior of the tertiary structure, while the hydrophilic amino acid residues are on the exterior. This causes haemoglobin to be soluble in water.

Terminology RNA polymerase: one strand of dsDNA used as template for ss-mRNA Ribosomes identify AUG initiating sequence t-RNA: anti-codon matched with codons on ss-mRNA corresponding amino acids bond to one another to form polypeptide, detach Amino acid residue = what s left of the amino acid after peptide bonds have been formed/ condensation polymerisation has occured Advantageous VS Deleterious mutations N-terminus = free amino group C-terminus = free carboxyl group Alpha helix from different angles R-groups pointing radially outwards Beta-pleated sheets, parallel and anti-parallel Amino acids determine whether a region is pleated, helical, or a kink Noncovalent, weak interactions are formed, such as Hydrogen bonds and Van der Waals interactions to achieve shape The diffusion collision model, in which a nucleus is formed, then the secondary structure is formed, and finally these secondary structures are collided together and pack tightly together The nucleation-condensation model, in which the secondary and tertiary structures of the protein are made at the same time Solubility a result of apolar amino acids facing inwards and polar facing outwards: allow dipoledipole interactions with solvent. Globular proteins consist mainly of enzymes, messenger proteins and transport proteins Increase/ Decrease fitness of organism/ person