Amino Acids and Proteins

Proteins are organic molecules consisting of many amino acids bonded together. General Properties o Most abundant biomolecule; accounts for 50% of dry weight. o Built by assembling long chains of amino acids (monomers), followed by intricate folding o Final shape of protein is very specific. Unless correctly folded, is not functional. o several 1000 different types of proteins in any cell; millions of protein molecules o To understand cellular life, must understand what different proteins are doing, how they work. A vast, but doable, challenge. Functions o rigid structure -collagen in connective tissue, bone; keratin in fingernails and hair; silk fibers o enzymes ; 3-D stereospecific chemical catalysts accelerate desired reactions by as much as 10 10 times over their spontaneous rates.. o transport : membrane transport proteins carry substances across cell membranes; blood transport proteins that move certain substances (e.g., iron, oxygen, cholesterol) throughout the body. o hormones ; chemical signals. Some hormones consist of as little as a single amino acid. Others are peptides or polypeptides. Example: insulin o contraction ; muscle fibers, cilia, spindle fibers in mitosis. o specific binding : e.g., that bind specifically to foreign substances to identify them to the body's immune system o buffers; proteins form zwitterions. The amino group accepts a + proton and becomes NH3 and the carboxyl group (COOH) donates a proton and becomes dissociated (COO-). The net charge on protein molecules varies with pH and is zero at the isoelectric point. Therefore the isoelectric point is important in considering the behaviour of food proteins because at this pH many properties are either at maximum or minimum. For example, electrical conductivity, solubility and viscosity are all a minimum. Electrical conductivity is minimum because net charge on each molecule is zero. Solubility is also minimum because there is a strong electrostatic attraction between neighbouring molecules hence molecules pack closely together. Hydrogen bonding also contribute to the close packing thus reducing the possibility of interaction with solvent molecules as a result, solubility msc 08-07 1 becomes minimum. Insolubility affects gel formation hence viscosity is also minimum. Amino acids with more amino groups than COOH are basic, and have isoelectric points above pH 7, while those with more COOH groups than amino are acid hence their isoelectric points are below pH 7.

Structure of Amino Acids o Each amino acid has an amino group and a carboxyl group, joined by a single Carbon atom. In addition, each amino acid has a characteristic "side chain" (often called the -R group). These determine the chemical properties of proteins. o Each amino acid (except glycine) can occur in two isomeric forms, L- and D-, because of the possibility of forming two different enantiomers (stereoisomers) around the central Carbon atom. Only L-amino acids are found in proteins in all organisms. o Note: some D-amino acids are found in bacterial cell walls. o "R" side group can be any of 20 different chemicals. o The most common amino acid, glycine, is only mildly nonpolar. o The great variety of side chains allows proteins to have many different (chemical) properties, and to create many different environments. For example; a. The presence of hydroxyls, carboxylates, sulfhydryls, and amino groups allows hydrogen bonding and the alkyl groups provide hydrophobic interactions, within the protein polymer itself and between separate protein molecules. b. Hydroxyl groups of serine, threonine and hydroxyproline can participate in ester linkages which have important influences on the properties of protein, as in the ability of enzymes to bind cofactors. c. The phenolic hydroxyl of tyrosine contributes to the acidic properties of proteins. d. Arginine yields a red colour when treated with - naphthol and sodium hypochlorite.

Peptides and Polypeptides a. Amino acids are not accumulated by cells, but quickly joined into specific assemblies by the formation of peptide bonds . b. The peptide bond that binds amino acids is one of the strongest and most durable of covalent bonds. In the laboratory, we can break, or hydrolyze, peptide bonds most effectively by a combination of heat and acid. In your body, this digestive process begins in the stomach, msc 08-07 2 where a combination of acid and enzymes help to break peptide bonds. If you didn't need to digest proteins, you wouldn't need a stomach! c. Two amino acids joined = dipeptide. Three AAs joined = tripeptide Many AAs joined = polypeptide . d. Short chains of amino acids with low molecular weight are called peptides. Polypeptide refers longer chains of amino acids (aprox. more than 30 amino acids). Every peptide and polypeptide has one free amino group (called the "N-terminus") and one free carboxyl group(called the "C-terminus"). " Protein " refers to the overall functional assembly, created when one or more polypeptides fold up and become functional units. Some proteins consist of only a single folded polypeptide chain, but many proteins contain multiple polypeptides, and frequently inorganic atoms as well, such as Zinc, Iron, Magnesium, etc.

Protein Structure 1. Protein structure is determined by several factors. These same factors determine the proteins folding, stability, solubility and purification, function and specificity, etc. a. Chemical composition o atoms, bonds, bond types = amino acids amino acid sequence, peptide bonds = primary sequence

b. Chemical conformation o side chain conformation o backbone conformation, restricted conformations o alpha helices, beta strands, phi and psi values, etc. o hydrogen bonding c. Chemical composition and the roles played by each type: o hydrophobic o aromatic o hydrophilic o hydrogen bonding o electrostatic and ionic

2. Amino acids link through peptide bonds to form polymers. o The polymers are called peptides (less than 30) or proteins (greater than 30). o The structures of the proteins are determined by the sequence of amino acids. o The possible structures of the backbone of the proteins are restricted by the peptide bond. The peptide bond is planar about the C, O, N and NH atoms. Due to the bulk of the side

msc 08-07 3 chain and other atoms the protein has a restricted conformation about the phi and psi bonds.

Levels of Structure 1 o Structure o Primary structure is the sequence of amino acids in the polypeptide chain. This represents the covalent bonding in the protein molecule. e.g. Val-Leu-Ser-Glu-Gly-Glu-Trp-Gln- Leu-Val- represents the first ten amino acids of myoglobin (out of 153 total). o Note: since every polypeptide begins with free amino group, this is called the N-terminus. The opposite end of the polypeptide has a free carboxyl group, called the C-terminus.

2 o Structure 1. Polypeptides fold in a series of stages. The first level of folding is called the secondary (2o) structure. Secondary structure is the organization of the polypeptide into regular repetitive patterns over short segments of 5-15 amino acids, for example the helical segments in the myoglobin structure.

2. The alpha-helix is one of the most common 2o folding patterns discovered by Pauling and Corey. o Alpha helix: Hydrogen bonds can form readily between C=O groups in the backbone and N-H groups four amino acid residues further along the chain. o This regular pairing pulls the polypeptide into a helical shape that resembles a coiled ribbon.

3. Another common folding pattern is called beta-pleated sheet. In this structure, polypeptides may run parallel to each other and hydrogen bonds are formed between a carbonyl group of one chain and the hydrogen of the imino group of another, thus linking the two-polypeptide chains together. The number of hydrogen bonds formed far much exceeds those in alpha-helix structure. Therefore the beta-pleated structure is much more stable. 4. Some protein regions remain in random coil, no regular pattern of secondary structure. 5. Different proteins have different degrees of alpha helix, beta sheet, and random coil. Silk is a protein stabilized entirely by pleated sheet; keratin (in hair) is stabilized entirely by alpha helix. Most proteins have some or both. 6. The nature of the hydrogen bonding confers stability onto the heling structure and the elasticity of such a molecule is due to the fact that hydrogen bonds can stretch much more than covalent bonds. The secondary structure accounts for the marked elasticity of some fibrous protein like keratin in wool or hair.

msc 08-07 4 7. The secondary structure is based on the rotationary angles assumed about the covalent bond interconnecting the amino acid and the peptide bond. This rotation occurs between; a. The N and the -Carbon atom (phi) b. -Carbon atom and the carbonyl carbon. (psi

3o Structure Tertiary structure is the overall folding of the whole polypeptide. o Polypeptides may continue folding beyond the formation of secondary structure. It is only with the complete, compact folding into tertiary (3 o ) structure that they attain their "native conformation" and become active proteins (as a result of the creation of active sites). o Forces that contribute to tertiary folding include: a. Hydrogen bonds b. Hydrophobic bonds - interactions between nonpolar regions. c. Ionic bond - between adjacent side chains. d. Sulfhydryl bonds (-S-S- bonds). These are especially important, because they are covalent bonds and quite strong compared to H-bonds.

4o Structure Some proteins are made of multiple polypeptide subunits, which must be assembled together after each individual polypeptide has reached its 3o structure. Examples: a. Hemoglobin (blood protein involved in oxygen transport) has four subunits.

b. Pyruvate dehydrogenase (mitochondrial protein involved in energy metabolism) has 72 subunits. Quaternary structure is the grouping of several protein molecules into a single larger entity; the subunits may act cooperatively with each other to give the grouping special properties not possessed by the single subunit. Not all proteins have a quaternary structure, e.g. a single myoglobin molecule functions by itself, but its close relative, hemoglobin, is a tetramer of four globin subunits. Each globin is structurally very similar to myoglobin. The bonding mechanisms that hold the protein chains together are generally the same as those involved in the tertiary structure with the exception of disulphide bonds that are considered unimportant in maintaining the quaternary structures.

msc 08-07 5 Conjugated Proteins Proteins may exist in combination with carbohydrates, lipids, nucleic acids, metal ions or phosphates. These prosthetic groups are bound to the protein by linkages other than salt linkages. The conjugation confers stability to the protein. For example o Lipoproteins: complexes with lipids o Glycoproteins: conjugated with heterosaccharides. o Metalloproteins: complexes with heavy metals o Nucleoproteins: protein and nucleic acid found in viruses and ribosomes. o Phosphoproteins: complexes with inorganic phosphate.

Ionic Properties of Proteins o Hydrophilic R-groups are generally located on the exterior of the protein molecule, while the hydrophobic or non-polar are in the interior. o Titration experiments show that nearly all of the potentially ionizable R-groups of the protein are capable of ionizing. o A protein with a high content of acidic groups amino acids (aspartic and glutamic) has a low isoelectric point. If it has more basic amino acid (arginine, lysine) it has a high isoelectric point. o If it binds ions of neutral salts, (magnesium & calcium) a change in both the isoelectric point and the titration curve occurs. o Variations in ionic properties pave way for several methods for fractionation proteins from biological systems to include, electrophoresis, ion-exchange chromatography. o Electrical charge also influences solubility. As the isoelectric point is approached the charge difference between molecules is lessened, hence proteins touch, aggregate and sometimes precipitate. o Proteins that are water-soluble owe their solubility to water protein hydrogen bonding and the environmental pH that do not coincide with their electric points. This background is used to precipitate milk casein. Ions with neutral salts react with charges on proteins decreasing the electrostatic attraction between opposite charges of neighboring protein molecules and increase in solvation and enhance their solubility. This is called 'salting in'. Precipitation occurs with an increase in the concentration of neutral salts - 'salting out'. o Organic solvents eg. acetone, ethanol, compete with the protein for water thus decrease the dielectric constant of the solution. This decreases the repulsive forces of like

msc 08-07 6 charges making the proteins aggregate and precipitate. This method can be used to fractionate protein from biological material. o Generally, solubility increases with temperatures between 0oC and 40oC except for (- casein which is soluble at 0oC. Above 40oC proteins denature thus lose solubility.

Denaturation & Renaturation o When proteins are heated, or exposed to acids or bases, or high salt concentrations, the variety of weak bonds holding tertiary and quaternary structure together can be disrupted so that the protein unfolds. Unfolding = denaturation resulting in loss of function. Unfolding can proceed even to disrupt secondary structure. Denaturation is therefore any modification of the secondary, tertiary or quaternary structure of a protein molecule, excluding, breakdown of covalent bonds (Fennema, O, 1985). o Denaturation is sometimes reversible; an unfolded protein can be restored to correct folding and regain biological activity. This is called renaturation, regeneration, reactivation. o Denaturation can also occur irreversibly (as when egg white protein, albumin, is denatured by boiling to congeal as egg white). Renaturation is then no longer possible. a. When denaturation occurs, peptide bonds of the protein are more readily available for hydrolysis by proteolytic enzymes, solubility is decreased, enzymatic activity is decreased or lost, intrinsic viscosity is increased and optical rotation of a solution is increased. Protein molecules unfold and become more asymmetrical exposing more hydrophobic residues, thus decreasing solubility and increasing viscosity. b. Sensitivity to denaturation agents varies with proteins. c. Denaturation results differ with conditions and agents even if the protein molecule is the same.

Denaturation can be brought about by; a. Controlling pH - This occurs most readily in the isoelectric point when the proteins are least stable eg. Caseinogens in milk. b. Heat causes protein to coagulate. Slowly coagulated protein is easily digested compared to rapidly done, which results in a hard solid mass. Protein coagulation is widely used to recover recombinant protein products, enzymes and in the production of common foods. Denaturation by heat treatment is affected by addition

msc 08-07 7 of mineral salts, which cause the protein to precipitate out of solution resulting in an insoluble salt.

c. Beating egg white brings about partial coagulation of ovalbumin resulting in a foam which can be further coagulated into a rigid structure by heating. Foaming occurs at isoelectric point when the egg is lest stable. Addition of an acid improves foaming.

Effects of denaturation o Peptide bonds become more readily available for hydrolysis by proteolytic enzymes o Solubility is decreased-the protein becomes more assymetrical exposing hydrogen groups hence the insolubility. o Enzyme activity is decreased or lost o Crystalisation is no longer possible o Intrinsic viscosity increased- structure becomes more assymetrical. o Optical rotation of the protein is increased.

Gelation - Like carbohydrates, proteins are used to form gels. Proteins are able to bind by hydration approx. 1g of water per 5g of dry protein. Some proteins form gels by immobilizing water within the network. This water is physically entrapped and can be expelled from the gel when stress is applied. Immobilized water does not flow freely from a gel. Proteins with a high degree of asymmetry are capable of forming 3 dimensional matrix by establishment of interprotein hydrogen bonding. This cross-linking makes it possible for the structure to hold water in an immobilized state. The gel shrinks when pH is changed to approach isoelectric point, expelling some of the immobilized water through synerisis.

Food Uses of protein o Proteins are used for their thickening, gelling, emulsifying and water binding properties in meats (sausages), bakery products, cheese, deserts and salad dressings. o They are used for their cohesive and adhesive properties in sausage making, pasta and baked goods. o Egg proteins are used for their foaming properties in desserts, cakes and whipped toppings. o Milk, egg and cereal proteins are used as fat and flavour binders in low fat bakery products. o Proteins are used for texture and palatability in bakery products

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