Sequence Of Amino Acids Determines Biology Essay

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Proteins are the most structurally complex and functionally sophisticated molecules. Proteins are the building blocks of cell and perform nearly all the cell's activities. It constitutes the major part of the cell dry mass. They are the most versatile macromolecules in living systems and play vital role in almost all biological mechanisms. "They are also referred to as molecular machines of the cell". (Lodish etal, 2008) They are the polymers of amino acids and are available in various shapes and sizes. The study of its three dimensional structure has revealed that the 3-D structure of a protein is determined by its amino acid sequence and that the functions of a protein is based on its structure. Proteins are long chin of sequence of 20 different amino acids, each linked to its neighbour through a covalent peptide bond. Proteins are polypeptides which has unique sequence of amino acids. Repeating sequence of atoms along the core of the polypeptide chain forms its backbone. On this polypeptide backbone are attached side chains that are not involved in making a peptide bond and give each amino acid its unique character. "20 different amino acids have 20 different side chains, on polar, aliphatic side chain includes Ala, Gly, Ile, Leu, Met, Val and Pro, Phe, Tyr and Trp have aromatic side chains and are hydrophobic. The polar, uncharged side chains include Asn, Cys, Gln, Ser, and Thr. The negatively charged (acidic) amino acids are Asp and Glu and the positively charged (basic) are Arg, His and Lys." (Nelson & Cox, 2000)

"Variation in the length of proteins, its sequence of amino acids, variations in the number of disulfide bonds or the attachment of small molecules or ions to their amino acid side chains determines their 3-D diversity." (Lodish, 2008)

The arrangement of atoms of a protein in the space is called its conformation. Proteins fold into a conformation of lowest energy or any structural state that can be achieved without breaking covalent bonds. "Proteins in any of their functional, folded conformations are called native proteins." (Nelson & Cox.2000).

Therefore, in this assignment we'll see how the sequence of amino acids in the primary structure of protein give rise to its final 3-D structure of protein and we'll see how the 3-D structures of the protein is related to its functions. We'll also see the diversity of proteins according to its structure and function. To look into these matters first let us look into the conformation of proteins which is an important factor determining its final 3-D structure.

Conformation of proteins

A change in conformation can only occur by rotation about single bonds. The nature of the covalent bonds in the polypeptide chain places constraints on the structure. "The peptide bond shows partial double-bond character that keeps the entire peptide group in a rigid planar configuration." (Nelson & Cox, 2000). The single bonds along the N-C and C-C bonds can rotate with bond angles φ and ψ respectively. "The requirement that no two atoms overlap, greatly reduces the possible bond angles in a polypeptide chain." (Nelson & Cox, 2000). This constraint and other steric interactions greatly reduce the possible 3-D arrangement of atoms. Protein configurations which have the maximum number of weak interactions are generally considered the most stable with lowest free energy. Apart from covalent disulfide bonds, the initial folding and subsequent stability of a polypeptide depend on number of weak forces (30-300 times weaker than typical covalent bond). But many weak forces acting in parallel can hold 2 regions of a polypeptide chain tightly together. "There are four types of weak bonds: Hydrogen bonds, electrostatic (ionic) bonds, Vander Waals interaction and most importantly the hydrophobic distribution of its polar and non polar amino acids." (Albert etal, 2008). Hydrophobic residues are mainly buried in the core of the protein, away from water, thus increasing the number of H-bonds within the protein molecule. The conformation of protein determines its primary structure. The primary structure gives rise to secondary structure. The secondary structure gives rise to tertiary and quaternary structure. Therefore protein structures naturally occur in a hierarchy.

Hierarchical structure of Proteins

The hierarchies of protein structure consist of:

1. Primary structure: is its linear arrangement of amino acids. The amino acids in a polypeptide are linked by amide bonds formed between the C=O (carboxyl) group of one amino acid and the N-H (amino) group of the next. "This linkage, called a peptide bond, has several important properties: 1. It is resistant to hydrolysis. 2. The peptide group is planar and rigid because the C=O-NH bond has resonant double bond character. 3. Each peptide bond has both H-bond donor (the N-H group) and H-bond acceptor (the C=O) group. H-bonding between these backbone groups is a prominent trait of protein structure. Finally, the peptide bond is uncharged, which allows proteins to form tightly packed globular structure having significant amounts of the backbone buried within the protein interior." (Berg, 2007). Primary structure gives rise to secondary structure of proteins.

2. Secondary structure of proteins: secondary structure are stable spatial arrangements of segments of polypeptide chain held together by H-bonds between backbone amide and carboxyl groups and often involving repeating structural patterns. The common secondary structures are α -helix, β-sheet and short U-shaped β turn. The term random coil applies to highly flexible portions of a polypeptide chain that have no fixed three dimensional structures. Secondary structure is stabilized by H-bonds and φ and ψ angles. (Berg, 2007). The secondary structure consists of:

a. The α helix: in a polypeptide segment folded into an α helix, the backbone forms a spiral structure in which the carboxyl oxygen atom of each peptide bond is hydrogen bonded to the amide hydrogen atom of the four residues farther along the chain. R groups of the amino acid residues protrude outward from the helical backbone. The repeating unit is a single turn of the helix, which extends about 5.4 A along the long axis. The amino acid residues in an α helix have conformations with ψ =-600, φ = -600, and each helical turn includes 3.6 amino acid residues. α Helix is usually right handed and makes maximum use of the hydrogen bonding. Five different kinds of constraints affect the stability of an α helix:

"i). The electrostatic repulsion or attraction between successive amino acid residues with charged R groups. ii). The bulkiness of adjacent R groups. iii). The interactions between amino acid side chains spaced four residues apart. iv). The occurrence of Pro and Gly residues, and v). The interaction between amino acid residues at the end of the helical segment and the electric dipole inherent to the α helix. Therefore, the tendency of a given segment of a polypeptide chain to fold up as an α helix depends on the identity and sequence of amino acid residues within the segment." (Nelson & Cox, 2000)

Fig. Structure of α helix of protein (Albert, 2008)

b. The β sheet: In the β conformation," the backbone of the polypeptide chain is extended into a zigzag. The zigzag polypeptide chains can be arranged side by side to form a structure resembling a series of pleats." (Nelson & Cox, 2000). H- Bonds are formed between adjacent segments of polypeptide chain. The adjacent polypeptide chains in a β sheet can be either parallel or antiparallel (having the same or opposite amino to carboxyl orientations respectively). The bond angles for β sheet conformations are φ -135 and ψ +135. (Nelson & Cox, 2000)

Fig. Structure of β sheet protein. (Albert, 2008)

c. β Turns: There are connecting elements that link successive rings of α helix or β sheet conformation. β Turns that connect the ends of two adjacent segments of antiparallel β sheet are common. Gly and Pro residues occur in β turns because Glysine is small and flexible and amine nitrogen of Proline readily assumes the cis-configuration." The structure is 1800 turn involving four amino acid residues with C=O of the first amino acid residue forming a H-bond with the N-H group of the fourth." (Nelson & Cox, 2000). The secondary structure leads to tertiary structure of proteins.

3. Tertiary structure of Protein: The overall 3-D arrangement of all atoms in a protein is referred to as the protein's tertiary structure. Tertiary structure is primarily stabilized by hydrophobic interactions between non polar side chains, together with H-bonds between polar side chains and peptide bonds. These stabilizing forces compactly hold together elements of secondary structure- helix, strands, turns and coils. Chemical properties of amino acid side chain help define tertiary structure. "Disulfide bonds between the side chains of cysteine residues covalently link regions of proteins, thus restricting the mobility of proteins and increasing the stability of their tertiary structure." (Nelson & Cox, 2000). Fig. Disulfide -bond formation (Albert, 2008)

Based on their tertiary structure proteins can be broadly classified into:

a. Fibrous Proteins: "are large, elongated, stiff molecules composed of many tandem copies of a short sequence that forms a single repeating secondary structure." (Becker etal, 2006). Fibrous proteins, which often aggregate into large multiprotein fibers that don't readily dissolve in water, usually play a standard role or participate in cellular movements. Eg. Silk fibroin, keratins of hair and wool, collagen (in tendons and skin) and elastin (in ligaments and blood vessels).

b. Globular Proteins: are generally water soluble, compactly folded structures, often but not entirely spherical. Whether a specific segment of polypeptide will form an α helix, or a β sheet depends on the amino acids present in that segment. " Eg. Leu, Met, Glu, and strong "helix formers" whereas Ile, Val, and Phe are strong "sheet formers". Both Gly and Pro, the only cyclic amino acids are "helix breakers" and are infact mostly responsible for the bends and turns in α helix which usually occur at the surface of a polypeptide." (Becker, 2006). Secondary structure gives rise to structural motifs.

Structural motifs are regular combinations of secondary and tertiary structure.

Certain combinations of α helix and β sheets have been identified in many proteins. "These units of secondary structure, called "motifs" consist of small segments of an α helix and/or β sheet connected to each other by looped regions of varying length." (Becker etal, 2006). Among the most commonly encountered motifs are the β - α - β motif and the hairpin loop and helix-turn-helix motifs, α-helix based coiled coil or heptad-repeat structural motif. The short segments that connect α helix and β sheets are called random coils.(Becker, 2006). Tertiary structures with structural motifs give rise protein domains.

Domains: distinct regions of protein tertiary structure are often called to as domains. There are two main classes of protein domains: functional and structural.

A functional domain is a region of a protein that exhibits a particular activity characteristic of the protein. Eg. Catalytic activity of a kinase domain that covalently adds a phosphate group to another molecule.

"A structural domain is a region of approximately 40 or more amino acids in length, arranged in a stable, distinct secondary or tertiary structure, which often can fold into its characteristic structure independently of the rest of the protein." (Lodish etal, 2008). Eg. Hemagglutinin, has a globular domain and a fibrous domain.

"The incorporation of domains as modules in different proteins in the course of evolution has generated diversity in protein structure and function." (Lodish etal, 2008). Homologous proteins which have similar sequences, structure and function, evolved from a common ancestor. They can be classed into families and super families. (Lodish, 2008). The tertiary structure of proteins along with protein motifs and domains give rise to quaternary structure of proteins.

4. Quaternary structure:

"Describes the number and relative position of the subunits on multimeric proteins (more than one protein)." (Lodish etal, 2008). Hemagglutinin is a trimer of three identical subunits and hemoglobin is a tetramer of two identical α subunits and two identical β subunits (Lodish, 2008)

Fig. Hierarchical structure of protein (Albert, 2008)

Denaturation of Proteins (As a basis to understand its 3-D structure)

Biologists have studied protein folding in a test tube by using highly purified proteins. Treatment with certain solvents, which disrupts the non covalent interactions holding the folded chain together, unfolds, or denatures a protein. Agents such as urea effectively disrupt proteins non covalent bonds. "In the presence of a large excess of β-mercaptoethanol, the disulfides (cysteine) are totally converted into sulfhydryls (cysteine)." (Berg etal, 2007). When a protein is converted into a randomly coiled peptide without its normal activity, it is said to be denatured. When the denaturing solvent is removed, the protein often refolds spontaneously, or renatures, into its original conformation. This indicates that the amino acid sequence contains all the information needed for specifying the three dimensional shape of s protein. "The dependence of conformation on sequence is especially significant because of the intimate connection between conformation and function." (Berg etal, 2007). Although a protein chain can fold into its correct conformation without outside help, in a living cell special proteins called molecular chaperones often assist in protein folding. (Berg etal, 2007).

Protein Diversity

Proteins are manufactured by our body in various shapes and sizes. Tertiary and quaternary structure of proteins adds to their diversity and range. According to Nelson & Cox in 2000, proteins motifs and domains are the basis for protein structural classification. Everyday thousands of proteins are formed, each with a unique shape and a particular function. Each protein has a unique sequence of amino acids in its primary structure which imparts it with a unique character and function. Most relevant proof that structure of proteins is related to its function comes from the denaturation of proteins. If the tertiary or quaternary structure of protein is destroyed, it loses its function. Even if there is a single mistake in coding the proper amino acids in the polypeptide chain, it renders the protein functionless and is related to various genetic diseases. "A misfolded protein appears to be the causative agent of a number of rare degenerative brain diseases in mammals". (Nelson & Cox, 2000: 196). The misfolding of normal prion protein of brain causes spongiform encephalopathy which makes the brain look like a sponge with numerous holes.

Therefore keeping in mind the relation between structure and function of protein, proteins can be classified into: (Nelson & Cox, 2000)

1. Structural protein: these are dominantly fibrous proteins which provide mechanical support for cells and tissues. Eg. Alpha keratin of hair, feathers, nails, wool, hooves, horns etc. "α-keratin consists of two right-handed α-helices intertwined to form a type of left- handed super helix called an α-coiled coil"(Berg etal, 2007). The left-handed supercoil changes the position of two right-handed α helices in a way that there are 3.5 amino acid residues per turn instead of 3.6, which enables the side chain bonding to form at every seven residues, forming the heptad repeats. α- keratins are rich in hydrophobic residues and are very strong structures. (Berg etal, 2007)

Another fibrous component of skin is the collagen where basic function is to provide strength. It is found in skin, bone, tendon, cartilage and teeth. The collagen helix is slightly different from α helix. Each molecule is rod shaped, about 3000Ǻ long and only 15Ǻ in diameter. 'It contains three helical polypeptide chains, each nearly 1000 residues long'. (Berg etal, 2007). It's also a coiled coil and the supercoiled twisting is right -handed. (Nelson & Cox, 2000)

Silk fibroin- produced by insects consists of antiparallel β sheets rich in Ala and Gly residues, which are soft, flexible filaments and provide strength to the structures they are in. (Nelson & Cox, 2000).

They illustrate the relationship between protein structure and biological functions.

2. Enzymes: Are biological catalyst, which speed up essential chemical reactions. Eg. Protein kinases catalyze phosphorylation and phosphatases catalyze dephosphorylation. GTP, ATP, Trypsin, pepsin and DNA polymerase are other examples. "Catalytic properties of protein are due to its ability to bind to other macromolecules and to small molecules and ions". (Lodish etal, 2008). In many cases binding induce a conformational change in the protein and thus influence its activity. The substance that is bound by the protein is called as ligand. The region of a protein that associates with a ligand is known as the ligands binding site. "The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can form non-covalent bonds only with certain ligand". (Becker, 2006)

3. Transport proteins: membrane transporters carry molecules or ions across the cells which are usually large globular proteins in their quaternary structure. Eg. Myoglobin and haemoglobin- which are heme proteins that has the ability to bind molecular oxygen. Myoglobin is a monomeric heme protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bond oxygen which is then used for metabolic purposes. It's a polypeptide consisting of 153 amino acids, and of protoporphyrin IX and a central iron atom. "About 70% of the main chain is folded into eight α helices and much of the rest of the chain forms turns and loops between helices." (Berg etal, 2007).

Hemoglobin- which transports oxygen molecules across the tissues has higher capacity to bind molecular oxygen than myoglobin. Each subunits of a haemoglobin tetramer has a heme prosthetic group identical to that described for myoglobin. The allosteric properties of hemoglobin hat results from its quaternary structure differentiate hemoglobin's oxygen binding properties from that of myoglobin. (Nelson & Cox, 2000)

4. Signaling protein: carry signals between or within cells which are usually hormones. Eg. Insulin, Adrenalin, etc. Insulin is produced by islets of Langerhans of the pancreas, especially by the β cells. It has 2 chains, A and B. A chain has 21 amino acid residues and B chain has 30 amino acid residues. The two polypeptide chains are joined by disulfide cross-linkages. Its tertiary structure is stabilized by cysteines and other weak interactions. Insulin, in its quaternary structure can form hexamers due to hydrophobic interactions. The insulin which is stored in the β cells is toroidal (doughnut-shaped) which is released into the bloodstream and signals the liver to convert the blood sugar into glycogen for storage. (Wikipedia, the free encyclopedia)

5. Receptor proteins: detects signals and transmit them within cell. Eg. Rhodopsin detects light I the eye. Rhodopsin is a G-protein - coupled receptor. It is present in the rod cells of the retina."Rhodopsin absorbs photons and initiates G-protein signal transduction processes that result in electrical signals processed by the brain." (Stenkamp etal, 2002). It is a membrane protein. It has eight helical segments. The length and the orientation of the helices differ greatly. "Helix III is the longest and passes through the center of the protein. Helix VIIsI is a short helical segment on the cytoplasmic side of the membrane surface. The short β strands are located on the extracellular side of the protein near the retinal binding site." (Stenkamp etal, 2002)

6. Channel proteins: are proteins which fold into a channel or pore within a membrane though which molecules and ions flow. Some β barrels form large transmembrane of channels. Eg. Porin in the plasma membrane of cells. The non polar interior of membranes limits the free diffusion of polar molecules. In most membranes various proteins serve as channels and pumps which regulate the movement of ions, metabolites and even water across the membrane. The outside of porin is covered with hydrophobic residues, whereas the center includes a water-filled channel lined with charged and polar amino acids. (Albert etal, 2008)

7. Motor protein: Generate movement in cells. Eg. Actin and myosin in muscles. "Monomeric actin called globular actin (G-actin) joins to form a long polymer called filamentous actin (F-actin)." (Nelson & Cox, 2000). The filamentous actin joins end to end to form long thin filaments. The globular actin in the thin filament can bind strongly to one myosin head group. Myosin has a thick filament which is connected to the thin actin filament by myosin head. The myosin heads slide along the actin filaments, drawing the thick filaments into the thin filaments and creating muscle contraction. (Nelson & Cox, 2000)