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APPENDICES I. THEORY PRACTICAL LESSON 1 SIMPLE PROTEINS. STRUCTURE AND FUNCTION Appendix 1a. Classification and structure of amino acids More than 300 different amino acids have been described in nature. However, only 20 of them are commonly found as constituents of mammalian proteins. [Note: These are the only amino acids that are coded by DNA, the genetic material in the cell.] Each amino acid (except of proline) has a carboxyl group, an amino group, and a distinctive side chain or radical ("R-group") bonded to the α-carbon atom (Figure 1.1 A). At physiologic pH (approximately pH = 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (-COO-), while the amino group is protonated (-NH3+). In proteins almost all of these carboxyl and amino groups are combined in peptide chain and, in general, are not available for chemical reaction except for hydrogen bond formation (Figure 1.1B). Thus, the nature of the side chains ultimately dictates the role an amino acid in a protein. It is, therefore, useful to classify the amino acids according to the properties of their side chains into: nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases). A. Amino acids with nonpolar side chains Each of these amino acids has a nonpolar side chain that does not bind or give off protons or participate in hydrogen or ionic bonds (see Figure 1.2). The side chains of these amino acids possess lipid-like properties and that promote hydrophobic interactions. 1. Location of nonpolar amino acids in proteins: In proteins being present in aqueous solutions, the side chains of the nonpolar amino acids tend to cluster together in the interior part of the protein molecule (Figure 1.4). This is due to the hydrophobicity of the nonpolar R-groups which act much like oil droplets that coalesce in water. The nonpolar side chain R-groups fill up the interior part of the protein molecule and support its three-dimensional form. At the same time, in proteins that are located in a hydrophobic environment, like membranes, the nonpolar side chain groups are located on the surface of the protein, interacting with the external lipid environment (see Figure 1.4). 2. Proline: The side chain of proline and its α-amino group form a ring structure. Thus proline differs from other α-amino acids containing an imino-group, instead of an amino group (Figure 1.5). The unique geometry of proline’s molecule contributes to the formation of the fibrous structure of collagen, and often interrupts the α-helices found in globular proteins. B. Amino acids with uncharged polar side chains These amino acids have neutral net charge at neutral pH although the side chains of cysteine and tyrosine can lose a proton at alkaline pH (see Figure 1.3). Serine, threonine, and tyrosine contain polar hydroxyl groups that can take part in hydrogen bond formation (Figure 1.6). The side chains of asparagine and glutamine, contain carbonyl and amide groups, that can also participate in hydrogen bonds. 1. Disulfide (-S-S-) bond: The side radical of α-amino acid cysteine contains a sulfhydryl group (-SH), being an important component of the active center of a great number of proteins. The -SH groups of two cysteines may oxidise with subsequent conjugation forming a dimer named cystine, which contains a covalent cross-link called a disulfide bond (-S-S-). 2. Side chain radicals as sites of reaction with other compounds: A number of αamino acids like Serine, threonine, and tyrosine contain a polar hydroxyl group (OH) that serves as a site of attachment for reactive groups such as inorganic phosphate group. Moreover, asparagine’s amide group, as well as hydroxyl group of serine or threonine, can act as a site of binding for oligosaccharide chains of glycoproteins. C. Amino acids with acidic side chains Two α-amino acids, aspartic and glutamic acids, are proton donors. At neutral pH the side chain radicals of these amino acids are fully ionized, containing a negatively charged carboxyl group (-COO-). Consequently, these amino acids are called aspartate or glutamate in order to indicate that these amino acids are negatively charged at physiologic pH (see Figure 1.3). D. Amino acids with basic side chains The side chain radicals of basic amino acids accept protons (see Figure 1.3). At physiologic pH the side radicals in lysine and arginine residues are positively charged. In contrast, histidine is slightly basic. Therefore, it is mainly uncharged at physiologic pH being a free amino acid. However, when histidine is incorporated into a polypeptide chain, its side radical can be either positively charged or neutral, depending on the ionic environment provided by the polypeptide chains of the protein. This important feature of histidine contributes to its role in the proteins’ function (e.g. hemoglobin). Abbreviations and symbols for the commonly occurring amino acids Each amino acid name has an associated three-letter abbreviation and a oneletter symbol (Figure 1.7). The one-letter codes are determined by the following rules: 1. Unique first letter: If only one amino acid begins with a particular letter, then that letter is used as its symbol. For example, I = isoleucine. 2. Most commonly occurring amino acids have priority: If a number of one amino acids begins with a similar letter, the most prevalent amino acid receives this letter as its symbol. For example, threonine is more prevalent in comparison to Tyrosine, so T is a symbol of threonine. 3. Similar sounding names: Someone -letter symbols remind the amino acid they represent by sound (e.g., F = phenylalanine (ph = [f]) or W= tryptophan ("tWyptophan" by Elmer Fudd). 4. Letter close to initial letter: In the case of remaining amino acids, their oneletter symbol is designated as close in the alphabet as possible to the initial letter of the acid. For example, the letter “B” designates Asx (aspartic acid or asparagine) being located near “A” in the alphabet. F. Optical properties of amino acids The α-carbon atom of each amino acid (except of glycine) is binded to four different chemical groups and, consequently, is optically active (chiral) carbon atom. The α-carbon atom in the glycine’s molecule has two hydrogen substituents (3 different groups instead of 4 for other amino acids). Thus, glycine is optically inactive. Amino acids with an asymmetric center at the α-carbon exist in two different optical forms, designated as D- and L-isomers, being mirror images of each other (Figure 1.8). These two forms are termed as stereoisomers, optical isomers, or enantiomers. All amino acids being present in mammalian proteins are of the Lisomers. At the same time, D-amino acids are present in bacterial cell walls proteins. Appendix 1b. Acid-Base Behavior of Amino Acids Every α-amino acid contains both basic amino (NH2) and acidic carboxyl (COOH) groups. Consequently, hydrogen (proton) transfer from acidic to the basic group leads to “zwitterion” formation (from German, where “zwitter” means “double”). This zwitterion is a salt containing both single positive and single negative charges at distinct functional groups. Taking into account the presence of both positive (+1) and negative (-1) charges in the zwitterion, its net charge is neutral (0). Actually, an amino acid exists in different forms, depending on the reaction of the current medium. When the reaction of the medium is near-neutral (pH ~ 6), alanine and other neutral amino acids exist in zwitterionic forms (A) without net charge (0). In zwitterionic form the carboxyl group is negatively charged being a carboxylate anion- (-COO-) whereas the amino group is positively charged being an ammonium cation (-NH3+). When the reaction of the medium becomes acidic (pH = 2 or even lower), the carboxylate anion binds a proton originating from the acidified media becoming neutral (carboxyl group). At the same time, amino acid has a net positive (+1) charge (form B). When the reaction of the medium becomes alkaline (pH > 10) the ammonium cation loses a proton (the latter reacts with hydroxyl groups originating from the medium forming a molecule of water) and the amino acid has a net negative (-1) charge (form C). Thus, alanine exists in one of three different forms depending on the pH of the medium solution. At the physiological pH of 7.4, neutral amino acids are present mainly in zwitterionic forms. The value of pH at which the amino acid exists in zwitterion (neutral charge) is called its isoelectric point, abbreviated as pI. The pI of neutral amino acids are generally equal to pH ~ 6. Acidic amino acids, having one more carboxyl group, have lower pI values (around 3). The three basic amino acids, which have one more amine group that is able to accept a proton, have higher pI values (around 7.6-10.8). Appendix Ic. Peptide Bond Formation During formation of a dipeptide, the amine (–NH2) group of one amino acid forms an amide bond with the carboxyl (-COOH) group of another amino acid. A proton (H+) originating from amine group and hydroxyl anion (HO-) from carboxyl group react leading to the formation of water (H2O) that is released into the medium. For example, reaction of the -COO- group of alanine with the –NH3+ group of serine forms a dipeptide with one new amide bond. The dipeptide has an ammonium cation (–NH3+) at one end of its chain and a carboxylate anion (-COO-) at the other: When two or more amino acids are joined together by amide bonds, forming large molecules called peptides and proteins. A dipeptide has two amino acids connected to each other by one amide bond. A tripeptide has three amino acids joined by two amide bonds: Molecules containing from 12 to 20 amino acid residues are called oligopeptides. The ones containing more than 20 amino acids are polypeptides. Proteins are macromolecules with molecular weight from 5000 and more consisting of one or more polypeptide chains. The amide bonds in peptides and proteins are called peptide bond. Individual amino acids are called amino acid residues. The names of residues are formed by replacing the suffix –ine or –ate with –yl. For example, glycine residue in the peptide chain is called glycyl while glutamate residue is called glutamyl. In the cases of asparagine, glutamine and cysteine, -yl replaces the final –e forming asparaginyl, glutaminyl, and cysteinyl, respectively. Every polypeptide chain has a number of universal structural properties. At the one end of the chain one free α-amino group is located. This end is called the amino terminal (N-terminal) end and this amino acid is named as the first amino acid. The other end of the polypeptide chain is the carboxy-terminal end (Cterminal), where a free α-carboxyl group which is contributed by the last amino acid is located. Appendix Id. Protein folding. Role of chaperones in protein folding New polypeptides are synthesized de novo in the cell by a translation complex including ribosomes, mRNA, and various factors. As the newly synthesized polypeptide chain is released from the ribosome, it folds into its three-dimensional shape. Folded proteins take up a low-energy state that makes the native structure more stable. In most cases the native conformation is reached in less than a second, indicating that folding is a very rapid process. Protein folding and stabilization depend on disulfide bonds and several noncovalent forces including the hydrophobic effect, hydrogen bonding, van der Waals interactions, and charge-charge interactions. The weakness of each noncovalent interactions allow proteins to undergo small conformational changes. This results in a correctly folded protein with a low energy state. When new proteins are not folded correctly, they may react with other proteins and consequently form aggregates. A number of neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, Huntington’s diseases, are caused by accumulation of protein deposits from such aggregates. Due to the high rapidity of protein folding this process should be determined by the primary structure of the polypeptide chain. The outcome of correctly folded proteins is up-regulated by a group of specialized proteins, molecular chaperones. Originally, the term “chaperone” was used for designation of an older person who accompanies the younger one(s) to ensure the right behavior. These proteins bind to the polypeptides before their folding is complete. It allows to prevent the formation of additional inter- and intramolecular bonds leading to incorrect folding and impaired protein structure. Moreover, chaperones may bind unassembled protein subunits preventing their conjugation before they are combined into a complete complex protein. A wide number of chaperones present in the living cell. However, most of them are heat shock proteins (HSPs) - proteins that are synthesized in response to an increase of temperature (heat shock) or other changes that cause protein denaturation in vivo. The role of HSPs is to repair the damage caused by denaturating factors by binding to denatured proteins and helping them to refold into their native conformation. Appendix Ie. Classification of proteins Peptide Classification Peptide is the term indicating short polymers of amino acids. Peptides are classified by the number of amino acids in the polypeptide chain. Each amino acid in the chain is called an amino acid residue, indicating the fragment left after the release of water resulting from the formation of peptide bond (Appendix Ic). Dipeptides have two amino acid residues, tripeptides – 3, tetrapeptides – 4, and so on. However, when the number of amino acid residues in the chain exceeds 12 but not 20 these polypeptides are called oligopeptides. If the peptide contains more than 20 residues, it is called polypeptide. Proteins Are Composed of One or More Polypeptide Chains For indication of single polypeptide chains both polypeptide and protein can be used. However, the term protein more often indicates a molecule composed of one or more polypeptide chains. The terms polypeptide and protein are used interchangeably in discussing single polypeptide chains. If the protein molecule has only one polypeptide chain, this protein is called monomeric protein. When there is more than one chain the protein molecule, it is called multimeric (di-; trimeric etc.) protein. If the structure of polypeptides in the protein is equal, this protein is called homomultimeric. Oppositely, when the structure of polypeptides in the protein molecule is distinct, this protein is heteromultimeric. Greek letters and subscripts describe the peptide composition of multimeric proteins. Thus, an α2-type protein is a dimer of identical polypeptide subunits, or a homodimer. Hemoglobin consists of four polypeptides of two different kinds; it is an α2β2 heteromultimer. Architecture of Protein Molecules Protein Shape All present proteins can be classified into one of three global classes on the basis of shape and solubility: fibrous, globular, or membrane. 1. Fibrous proteins have relatively simple, regular linear structures. These proteins carry out structural function in cells. Commonly, they are not soluble in aqueous solutions. 2. Globular proteins have spherical shape. They are folded so that hydrophobic amino acid side chains are located in the interior of the molecule in order to avoid contact with water. Hydrophilic side amino acid residues are located on the surface of the molecule contacting with water. Consequently, globular proteins are characterized by good solubility in water. Cytosolic enzymes being soluble are globular in shape. 3. Membrane proteins are structurally associated with biological membranes. Hydrophobic amino acid residues are located inside the membrane for interaction with nonpolar phase presented by lipids. In this connection, membrane proteins are insoluble in aqueous solutions. However, in experimental procedures these proteins may be solubilized with various detergents. Membrane proteins in comparison with cytosolic proteins have less hydrophilic amino acids. The latter are located outside the membrane (Figure 1.4; Practical lesson I). Biological Functions of Proteins Proteins are biologically active agents. Proteins take part in all metabolic processes inside the cell. That is why one of the proteins classifications is based on their biological function. 1. Enzymes Enzymes represent the widest class of proteins (more than 3000 individual enzymes in the Enzyme Nomenclature and Enzyme Classification). Enzymes act as biological catalysts that accelerate the rates reactions inside the living organism. Each Enzymes are specific in their action and modulate individual metabolic reactions (Practical lesson 3). Virtually every step of the metabolism is catalyzed by one or more enzymes. The catalytic activity of enzymes manifold exceeds the one observed for inorganic catalysts. Enzymes enhance the reaction rates to maximum of 1016-fold in comparison to the spontaneous uncatalyzed reaction. Enzymes are classified and named according to the type of the reaction they catalyze (glutathione transferase (GST) transfers glutathione into the acceptor molecule, whereas glutathione reductase (GR) catalyses reduction of oxidized glutathione into reduced glutathione). The systematic names of enzymes come from the type and the participants of the reaction they catalyze. For example, the name “glyceraldehyde-3-phosphate:NAD oxidoreductase” means that the substrate of the reaction is glyceraldehyde-3-phosphate, the reaction type is reduction/oxidation reaction in presence of the cofactor NAD. At the same time, the enzymes have also common names being less massive. For example, the above mentioned enzyme is also known as “glyceraldehyde-3-phosphate dehydrogenase”. A number of enzymes have trivial names with historical origin. For example, an antoxidant enzyme catalyzing decomposition of hydrogen peroxide catalase is systematically called as hydrogen-peroxide:hydrogen-peroxide oxidoreductase. 2. Regulatory Proteins A number of proteins do not catalyze obvious chemical reactions, however, they regulate the ability of other proteins to carry out their physiological functions. These proteins are called regulatory proteins. For example, insulin (from latin “insula” – an islet), a pancreatic hormone being a protein (5.7 kD), consists of two polypeptide chains (21 and 30 amino acid residues) connected to each other by disulfide bods (cross-bridges). Other proteins take part in regulation of gene expression. They bind to certain DNA sites activating or inhibiting the transcription of genetic information from DNA to RNA. For example, repressor proteins (or repressors) block the transcription process being negative regulators of transcription. Positive peptide regulators of transcription also present in the living cell. 3.Transport Proteins Transport proteins represent the third functional class of proteins. These proteins transport a wide number of chemical substances from one site of the cell or an organism to another. The most obvious example is hemoglobin that transfers oxygen from lungs to other tissues. Another type of transport is accompanied by the transport of different substances across biological membranes mediated by specific proteins. Membrane transport proteins (or in some cases just “membrane transporters”) transfer chemical compounds from the one side of the membrane to another. For example, family of glucose transporter (GluT) proteins transfer glucose from blood into the cell through cellular membrane. 4.Storage Proteins A number of proteins provide reservoirs for different chemicals (most often the latter are essential) being storage proteins. Obviously, proteins are reservoirs of nitrogen (e.g. ovalbumin or egg albumin). At the same time, other proteins store essential trace elements like iron in the case of ferritin. The latter is able to bind and store up to 4500 iron atoms. 5.Contractile Proteins A number of proteins allow cells to move in the medium. The contractile and motile proteins have a number of common properties. These proteins are filamentous or polymerize to form filaments. For example, actin and myosin interact forming contractile systems of the cell. Motor proteins like kinesis drive the movement of certain organelles. 6.Structural Proteins An important role of proteins consists in formation of the biological structures. These proteins also polymerize generating long and extra-long fibers. For example, collagen is an insoluble fibrous protein being a structural part of connective tissues, bones, forming dense extra-strong fibrils. Oppositely, elastin is a protein with elastic properties. It is an important component of elastic connective tissues. 7.Scaffold Proteins (Adapter Proteins) Scaffold or adapter proteins proteins play a significant role in the complex pathways of cellular response to hormones and growth factors. Adaptor proteins contain a large variety of protein-binding modules that link protein-binding elements together and facilitate the creation of larger signaling complexes. Generally, these proteins enhance protein-protein interactions leading to the formation of protein complexes. 8.Anchoring (targeting) proteins bind other proteins leading to their association with other cellular structures. , causing them to associate with other structures in the cell. Particularly, a family of anchoring proteins, known as AKAP or A kinase anchoring proteins, exists in which specific AKAP members bind the regulatory enzyme protein kinase A (PKA) to particular subcellular compartments. 9.Protective and Exploitive Proteins A number of proteins are protective and exploitive because of their role in defense and protection. The most expressed defensive action is characteristic for immunoglobulins that recognize and neutralize foreign bacterial, viral and other antigens without affecting cells and tissues of the host organism. Simple proteins and conjugated proteins Many proteins consist of polypeptide chain(s) and contain no other chemical groups. Such proteins are called simple proteins. At the same time, many other proteins contain various chemical constituents as an integral part of their structure. These proteins are termed conjugated proteins. If the nonprotein part is strongly binded to the protein’s molecule even by covalent bonds, it is called a prosthetic group. If the nonprotein component is not covalently linked to the protein molecule, it is called coenzyme. Сonjugated proteins are classified according to the chemical nature of nonprotein part. 1.GLYCOPROTEINS. Glycoproteins are proteins that contain a carbohydrate fragment in their structure. Proteins located extracellularly are often glycoproteins. For example, proteoglycans are important components of the extracellular matrix. Moreover, many transmembrane proteins are glycosylated on their extracellular sites. 2. LIPOPROTEINS. Lipoproteins are protein molecules containing lipid component in the structure. The most representative examples of this class of proteins are blood plasma lipoproteins. These lipoproteins act as lipid transporters from the site of synthesis to target cells and tissues. The ratio between different serum lipoprotein particles especially low-density lipoproteins (LDLs) and highdensity lipoproteins (HDLs) indicate the risk of atherosclerosis. 3. NUCLEOPROTEINS. Protein and nucleic acid conjugates termed nucleoproteins have many roles in the storage and transmission of genetic information. 4. PHOSPHOPROTEINS. Phosphoproteins are characterized by the presence of phosphate groups binded to hydroxyls of serine, threonine, or tyrosine residues. Regulation of protein active or inactive state is realized through phosphorylation (connection of phosphate group) of its center. 5. METALLOPROTEINS. Metalloproteins are proteins that contain metal ions (most often transition metal ion) in their active center. In the case of enzymes, these enzymes are called metalloenzymes. At the same time, metal-storage proteins like ferritin also may be classified as metalloproteins. 6. HEMOPROTEINS. Hemoproteins contain heme as a prosthetic group in their structure. The most obvious example of this class is hemoglobin. 7. FLAVOPROTEINS. Flavoproteins are chromoproteins (“coloured proteins”) containing the derivatives of flavin, FMN and FAD, as prosthetic groups. Appendix If. PRACTICAL LESSON 5 WATER-SOLUBLE AND LIPID- SOLUBLE VITAMINS. VITAMINS AS COFACTORS Nomenclature and classification of vitamins Vitamins are chemically unrelated organic compounds that being essential cannot be synthesized by humans and, therefore, must be supplied by the diet. Nine vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin, riboflavin, biotin, and pantothenic acid) are classified as water-soluble, whereas four vitamins (vitamins A, D, E and K) are termed lipid-soluble. Vitamins are required for specific cellular functions. For example, many of the water-soluble vitamins are precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to the water-soluble vitamins, only one fat soluble vitamin (vitamin K) has a coenzyme function. These vitamins are released, absorbed and transported with dietary lipids. They are not readily excreted in the urine, and significant quantities are stored in the liver and adipose tissue. In fact, consumption of vitamins A and D in excess of the recommended dietary allowances can lead to accumulation of toxic quantities of these compounds. A vitamin-deficient state (disease) can occur when a vitamin is deficient or absent in the diet. Such diseases can be treated or prevented by consumption of the respective vitamin. Classification of the Vitamins