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4/2/2012 Introduction Proteins • Proteins are essential parts of organisms and participate in every process within cells. • Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. • Through the process of digestion, animals break down ingested protein into free amino acids that are then used in metabolism. Red blood cells contain the oxygen transporting protein hemoglobin 1 Aspects of a protein's structure 2 Primary structure of proteins • There are four distinct aspects of a protein's structure: • Primary structure, • Secondary structure, • Tertiary structure and • Quaternary structure. • This is the amino acid sequence • An amino acid exists as a dipolar salt H H H2N C COOH H3N C COO R R R = alkyl • All amino acids have this general structure 3 4 1 4/2/2012 Classification of amino acids Anatomy of an amino acid • Non-polar with aliphatic R groups Glycine Alanine Proline Valine 5 Aromatic R groups Non-polar with aliphatic R groups Leucine 6 Isoleucine Phenylamine Methionine 7 Tyrosine Tryptophan 8 2 4/2/2012 Polar Uncharged R groups Serine Threonine Positively charged R groups Cysteine Arginine Lysine Asparagine Glutamine 9 10 The origin of the single-letter code for the amino acids Negatively charged R groups Aspartate Histidine Glutamate 11 Amino acid 3 letter Single letter Comment Histidine Isoleucine Methionine Serine Valine Alanine Glycine Leucine Proline Threonine His Ile Met Ser Val Ala Gly Leu Pro Thr H I M S V A G L P T These amino acids occur more frequently in proteins than do the other amino acids having the same first letters. 12 3 4/2/2012 Some of the other amino acids, are phonetically suggestive Amino acid 3 letter Single letter Arginine Phenylalanine Tyrosine Tryptophan Aspartic acid Asparagine Glutamic acid Glutamine Lysine Arg Phe Tyr Trp Asp Asn Glu Gln Lys R F Y W D N E Q K Summary: Non-polar amino acids Comment Twyptophan asparDic asparagiN glutamEke Q-tamine (K is near L in alphabet) 13 Summary: Polar, non-charged amino acids 14 Summary: Negatively-charged amino acids 15 16 4 4/2/2012 Formation of a peptide bond Summary: Positively-charged amino acids • Amino acids are linked by PEPTIDE BONDS which are covalent in nature • Peptide bond is an amide linkage formed by a condensation reaction (loss of water) • Brings together the alpha-carboxyl of one amino acid with the alpha-amino of another • Portion of the amino acid left in the peptide is termed the amino acid RESIDUE – Amino acids sometimes called RESIDUES • R groups remain UNCHANGED – remain active • N-terminal amino and C-terminal carboxyl are also available for further reaction 17 Amino acid residue 18 Formation of a peptide bond • Definition of amino acid residue: – an amino acid molecule that has lost a water molecule by becoming joined to a molecule of another amino acid. H A peptide bond contains 19 C N O group 20 5 4/2/2012 Write the three-letter abbreviations for the following tetrapeptide: Example of a pentapeptide Ser-Gly-Tyr-Ala-Leu CH3 CH3 CH3 O S CH CH3 SH CH2 CH2 O CH2 O CH2 O NH3 CH C N CH C N CH C N CH C O H H H Ala-Leu-Cys-Met **This is not the same as Met-Cys-Leu-Ala** 21 INSULIN 22 Insulin • Insulin has 51 amino acids, divided between two chains. One of these, the A chain, has • 21 amino acids; the other, the B chain, has 30. The A and B chains are joined by disulfide • bonds between cysteine residues (Cys-Cys). 23 24 6 4/2/2012 Secondary structure of proteins • This is the regularly repeating local structures stabilized by hydrogen bonds • Hydrogen bonds are electrostatic interactions between a donor consisting of the dipole of a polar O-H or N-H bond and an acceptor, consisting of an available lone pair of electrons on a neighbouring N or O atom. • Typical hydrogen bonds are about 5 - 10% as strong as a normal covalent bond, and are not permanent bonds like covalent bonds. • The dashed line - - - represents the hydrogen bond. Typical H-bond donors N-H - - -:N typical H-bond acceptors N-H - - -:O O-H - - -:N O-H - - -:O 25 Hydrogen bonds in secondary structures of proteins 26 Secondary structure of proteins: a-helix • Although each hydrogen bond is relatively weak in isolation, the sum of the hydrogen bonds in a helix makes it quite stable. • The H-bonds result in a strong but temporary attraction between H-bonding partners. 27 28 7 4/2/2012 Secondary structure of proteins: a-helix An α -helical secondary structure. • Hydrogen bonds between ‘ backbone ’ amide NH and C= O groups stabilize the α -helix. • Hydrogen atoms of OH, NH or SH group (hydrogen donors) interact with free electrons of the acceptor atoms such as O, N or S 29 Hydrogen bonds in secondary structures of proteins : b-pleat 30 The parallel β -sheet secondary structure. • If the H-bonds are formed between peptide bonds in different chains, the chains become arrayed parallel or antiparallel to one another in what is commonly called a β -pleated sheet. • That is: When the zigzag polypeptide chains are arranged side by side, they form a structure resembling a series of pleats. • The β -pleated sheet is an extended structure as opposed to the coiled α -helix. • It is pleated because the carbon—carbon (C—C) bonds are tetrahedral and cannot exist in a planar configuration. 31 32 8 4/2/2012 • In an antiparallel arrangement, the successive β-strands alternate directions of the N and Cterminus. This is the most stable β-sheet arrangement. • In a parallel arrangement, the N-termini of successive strands are oriented in the same direction, generating a less stable β-sheet due to the non-planarity of the inter-strand Hbonds. 33 34 Examples of amino acid side chain interactions contributing to tertiary Structure Tertiary Structure of Proteins • T he three-dimensional, folded and biologically active conformation of a protein is referred to as its tertiary structure. • This structure reflects the overall shape of the molecule. • T he three-dimensional tertiary structure of a protein is stabilized by interactions between side chain functional groups: covalent disulfide bonds, hydrogen bonds, salt bridges, and hydrophobic interactions. 35 36 9 4/2/2012 Stabilizing interactions responsible for the tertiary structure of a protein. Tertiary Structure of Proteins • These complex structures is held together by a combination of several molecular interactions that involve the R-groups of each amino acid in the chain. • These interactions include – – – – hydrogen bonds between polar R- groups ionic bonds between charged R-groups hydrophobic interactions between non-polar R-groups covalent bonds: The disulfide bond 37 38 Tertiary Structure of Proteins… Tertiary Structure of Proteins… • The importance of disulfide bonds in the structure of certain proteins is demonstrated by hair. Hair is made of the protein apha-keratin. The particular structure of your hair (straight, curly, etc.) is based on specific disulfide bonds that naturally form in the hair protein. This should help explain why an individual with straight hair cannot simply heat their hair, denature the protein (keratin), put in curlers and make it curly. The disulfide bonds are covalent bonds and thus are very strong. Heating these bonds will not break them, so simply heating hair will not change straight hair to curly. • Instead, it is necessary to break these bonds chemically, reform the hair to the desired shape, and make new disulfide bonds to maintain the new shape. • If an individual goes to the hairdresser for a permanent, the beautician must first treat the hair with a reagent that reduces (and thus breaks) the disulfide bond, then put in curlers (to get the desired shape), and add an oxidizing agent to form new disulfide bonds to maintain the new shape. 39 40 10 4/2/2012 Tertiary structures are quite varied Tertiary structure is important! The function of a protein (except as food) depends on its tertiary structure. If this is disrupted, the protein is said to be denatured and it loses its activity. For example: • denatured enzymes lose their catalytic power • denatured antibodies can no longer bind antigen A mutation in the gene encoding a protein is a frequent cause of altered tertiary structure. 41 Quaternary structure of proteins 42 Examples of quaternary structures Tetramer Hexamer SSB Allows coordinated DNA binding DNA helicase Allows coordinated DNA binding and ATP hydrolysis Filament • It is the shape or structure that results from the interaction of more than one protein molecule, usually called protein subunits, which function as part of the larger assembly or protein complex. 43 Recombinase Allows complete coverage of an 44 extended molecule 11 4/2/2012 Different structures of proteins • Some proteins (such as hemoglobin) have more than one peptide chain (these are multimeric proteins). The manner in which these chains fit together is the quaternary structure. • The subunits of a multimeric protein may be identical (homomultimeric protein), homologous or totally dissimilar (heteromultimeric protein ) and dedicated to disparate tasks. In some protein assemblies, one subunit may be referred to as a "regulatory subunit" and another as a "catalytic subunit." • The protein hemoglobin is made up of four polypeptide chains, two apha chains and two beta chains 45 46 Major Classes of proteins • The number of subunits in an oligomeric complex is described using names that end in –mer (Greek for "part, subunit") • 1 = monomer, 2 = dimer, 3 = trimer, 4 = tetramer, 5 = pentamer 6 = hexamer, 7 = heptamer, 8 = octamer, 9 = nonamer, 10 = decamer, 11 = undecamer, 12 = dodecamer etc • Although complexes higher than octamers are rarely observed for most proteins, there are some important exceptions: A capsid is the protein shell of a virus. It consists of several oligomeric (e.g. 60) structural subunits made of protein called protomers. Protein types • Proteins fall into three general classes, based on their overall three-dimensional (tertiary) structure and on their functional role: – fibrous, – membrane, – globular 47 48 12 4/2/2012 Fibrous Proteins Fibrous Proteins • Fibrous proteins tend to be long, narrow molecules. Fibrous proteins are used to construct macroscopic structures, especially structures outside of cells. Fibrous proteins tend to have a structural role, although some have more active functions as well. • Fibrous proteins are elongated molecules in which the secondary structure (either a-helices or b-pleated sheets) forms the dominant structure. • Fibrous proteins are insoluble, and play a structural or supportive role in the body, and are also involved in movement (as in muscle and ciliary proteins). • One feature of fibrous tissues is that they often have regular repeating structures. 49 • Keratin, for example, which is found in hair, horns, wool, nails, and feathers, is a helix of helices (2 pairs of a-helices wound around one another) and has a seven amino acid repeating structure. Other keratins are found in skin, fur, hair, wool, claws, nails, hooves, horns, scales, beaks, feathers, actin and mysin in muscle tissues and fibrinogen needed for blood clots. • Fibroin is the fibrous protein that makes up silk cloth and spider webs. • Silk is a fibrous protein that is composed only of bsheets. It too has a repeating pattern: layers of glycine alternate with layers of alanine and serines in the b-sheets. • Collagen, is the major protein component of connective tissue. In collagen, every third amino acid is glycine and many of the others are proline. • fibrous proteins generally have only primary and secondary structure whereas 50 Globular Proteins Membrane proteins • Globular proteins are by far the most abundant class of proteins. Many of the most heavily studied proteins are members of this class of proteins. • They are a highly diverse group of proteins that are soluble and form compact spheroidal molecules in water. • All have tertiary structure and some have quaternary structure in addition to secondary structure. Regular secondary structures generally comprise less than half the average globular protein. • Globular proteins typically consist of relatively straight runs of secondary structure joined by stretches of polypeptides that abruptly change direction. • Major examples include insulin, hemoglobin, most enzymes, transport proteins and receptor proteins. • Membrane proteins typically have a hydrophobic region (frequently α-helical) that interacts with the non-polar interior of membranes. • Membrane proteins often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane. 51 52 13 4/2/2012 Membrane proteins • A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle (a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer - a thin membrane made of two layers of lipid molecules). • Examples of organelles include: chloroplasts (in plants, algae etc), mitochondria (in almost all eukaryotes i.e. one of the structurally complex cell types) and cell nucleus • Example of a membrane protein 53 Some terminology 54 Cellular functions of proteins • Cell contains genome = complete set of DNA • Genes = specific sequences that encode instructions for making proteins • Protein = molecules of (20) amino acids that perform much of life’s function • Proteome = set of all proteins in a cell • Proteins are the chief actors within the cell, said to be carrying out the duties specified by the information encoded in genes • The set of proteins expressed in a particular cell or cell type is known as its proteome 55 56 14 4/2/2012 Enzymes Enzymes … • The best-known role of proteins in the cell is as enzymes, which catalyze chemical reactions. • Enzymes are usually highly specific and accelerate only one or a few chemical reactions. • They carry out most of the reactions involved in metabolism, as well as manipulating DNA in processes such as DNA replication, DNA repair, and transcription. • The molecules bound and acted upon by enzymes are called substrates. • Although enzymes can consist of hundreds of amino acids, it is usually only a small fraction of the residues that come in contact with the substrate, and an even smaller fraction - 3-4 residues on average - that are directly involved in catalysis. • The region of the enzyme that binds the substrate and contains the catalytic residues is known as the active site. 57 58 Cell signaling and ligand binding of proteins Structural proteins • Many proteins are involved in the process of cell signaling. • Antibodies are protein components of adaptive immune system whose main function is to bind antigens (foreign substances in the body) and target them for destruction. • Receptors and hormones are highly specific binding proteins. • Structural proteins confer stiffness and rigidity to otherwise-fluid biological components • Most structural proteins are fibrous proteins; for example, actin and tubulin are globular and soluble as monomers, but polymerize to form long, stiff fibers that comprise the cytoskeleton, which allows the cell to maintain its shape and size. 59 60 15