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Proteins Dr. Sarah Doyle School of Biochemistry and Immunology Lecture 2 Protein conformation The Genetic Code The Genetic Code: Genes are made up of sets of 3 nucleotides (codons) which encode a sequence of amino acids that form a polypeptide DNA codes for RNA (according to the base pair rule) RNA codes for protein: 3 nucleotides encode 1 amino acid: Protein: A sequence of amino acids form a polypeptide The genetic code ???How many different proteins are there in a human cell??? -‐ Latest es@mate: ~25,000 different proteins occur in humans -‐ How many proteins are needed to make a single cell? -‐ Different for different species: -‐ Worm (nematode): 19,000 -‐ Fly (fruit fly): 16,000 -‐ Yeast: 5,000 -‐ Mycobacteria: 500 -‐ Hepa@@s C virus: 8 (uses cell it infects) What is the structure of a protein? • Each of the ~25,000 human proteins have specific structure and function • Protein shape is very important to its function proteins interact with other proteins Eg. antibody • Diverse functions = diverse structures • Unique function = unique 3D shape • Protein Shape=Conformation Protein stuctures Polypeptides • Proteins – Are polymers of amino acids (aa) aa-aa-aa-aa-aa-aa-aa-aa-aa-aa-aa-aa-aa-aa polymer of aa = polypeptide • A protein – Consists of one or more polypeptides polypeptide(s) = protein • 1949: Fred Sanger determined the sequence of insulin Amino Acid Monomers • Amino acids – Are organic molecules possessing both carboxyl and amino groups – Differ in their properties due to differing side chains, called R groups R= reactive group Backbone of each aa – 20 different R groups = 20 different amino acids Amino Acid Monomers Unionised form Zwitterion form • internal transfer of a H ion from the -COOH to the -NH2 group = an ion with both a negative charge and a positive charge = over electrically neutral 4 subgroups of Amino acid -‐ There are 20 naturally-‐occurring amino acids -‐ Grouped into 4 subgroups, depending on the R-‐group: -‐ Non-‐polar: hydrophobic -‐Polar + Neutral: hydrophilic -‐ Polar + Acidic: hydrophilic hydrophilic hydrophobic -‐ Polar + Basic: hydrophilic A Polar compound is one where the spread of charge is not even, with some atoms carrying a partial negative charge and others a partial positive charge 1. Non-polar amino acids hydrophobic / water insoluble 2. Polar amino acids hydrophilic / water soluble Electrically charged amino acids water soluble 3. Acidic 4.Basic Amino acid abbreviations Non-polar Polar AMINO ACID glycine THREE LETTER CODE Gly SINGLE LETTER CODE G alanine Ala A valine Val V leucine Leu L isoleucine Ile I methionine phenylalanine Met Phe M F tryptophan Trp W proline Pro P AMINO ACID serine threonine cysteine tyrosine asparagine glutamine THREE LETTER CODE Ser Thr Cys Tyr Asn Gln SINGLE LETTER CODE S T C Y N Q Neutral AMINO ACID aspar@c acid glutamic acid THREE LETTER CODE Asp Glu SINGLE LETTER CODE D E Acidic AMINO ACID lysine arginine his@dine THREE LETTER CODE Lys Arg His SINGLE LETTER CODE K R H Neutral Basic Essential amino acids -those amino acids that an organism can not synthesize on its own Essen@al Nonessen@al Isoleucine Alanine Leucine Arginine* Lysine Aspartate Methionine Cysteine* Phenylalanine Glutamate Threonine Glutamine* Tryptophan Glycine* Valine Proline* His@dine Serine* Tyrosine* Asparagine* Obtained from diet * Conditionally essential, for some individuals, eg tyrosine in PKU Phenylketonuria (PKU) • Classical PKU is caused by a mutated gene for the enzyme phenylalanine hydroxylase (PAH) • PAH converts the essential amino acid phenylalanine to other essential amino acids eg tyrosine. • Lack of PAH causes phenylalanine accumulation, lack of tyrosine – normal at birth with gradual loss of mental function, severe learning disabilities and seizures • Treatment: intake of phenylalanine low, supplement diet with tyrosine Heel prick “Guthrie test” Amino Acid Polymers • Amino acids - Are linked by peptide bonds Peptide bond OH OH SH CH2 CH2 H H N Peptide bond is formed by a dehydration reactionCatalytic reaction CH2 H C C H O N H C C H O OH H (a) N C C H O OH H 2O OH CH2 Peptide bond - covalent bond H H H N (b) C C H O Amino end (N-terminus) Side chains SH Peptide CH2 bond CH2 OH N H C C H O N C C H O Carboxyl end (C-‐terminus) OH Backbone Protein Conformation and Function • Polypeptides - formed one at a time starting from N-terminus - range from a few monomers to 1000 or more • Specific polypeptides- unique sequence of aa’s (as determined by the genetic code) • Sequence of the aa polymer determines the 3D shape of the polypeptide • Proteins are not just chains of aa’s, they are defined by their shape – interactions between backbone residues and R-groups • A protein’s specific conformation - determines how it functions eg. Enzyme binds substrate The function of a protein is inextricably linked to its shape hemoglobin IgG Adenylate kinase insulin Glutamine synthetase Molecular surface of several proteins showing their compari@ve sizes • Two models of protein conformation Groove Eg. Lysozyme- an enzyme that Breaks down bacterial cell walls by recognizing and binding to specific molecules on the bacteria (a) A ribbon model Groove (b) A space-filling model Four Levels of Protein Structure • Primary structure – Is the unique sequence of amino acids in a polypeptide HN Amino acid + Gly Pro Thr Gly Thr Gly 3 Amino end subunits Glu CysLysSeu LeuPro Met Val Lys Val Leu Asp AlaVal Arg Gly Ser Pro Ala aa sequence determined by inherited genetic information Glu Lle Asp Thr Lys Ser Lys Trp Tyr Leu Ala Gly lle Ser ProPheHis Glu Ala Thr PheVal Asn His Ala Glu Val Thr Asp Tyr Arg Ser Arg Gly Pro Thr Ser Tyr lle Ala Ala Leu Leu Ser Pro SerTyr Thr Ala Val Val Glu ThrAsnProLys o c – o Carboxyl end Eg lysozyme is 129aa long • Secondary structure – Is the folding or coiling of the polypeptide into a repeating configuration – Includes the α helix and the β pleated sheet – Result of Hydrogen bonding between the repeating backbone of a polypeptide β pleated sheet O H H C C N Amino acid subunits C N H R R O H H C C N C C N O H H R C C N OH H R O R C H R O C O C N H N H N H O C O C H C R H C R H C R H C R N H O C N H O C O C H C O N H N C C R R C C H R O H H C C N C C N OH H R O C H H H C N HC C C N HC N N H H C O C C O R R O N O H H C C N R R H R O C H H NH C N C H O C R R C C O R H C N HC N H O C H α helix -Repeated coils or folds in patterns contribute to the overall conformation of a protein Hydrogen bonds • Hydrogen bonds are formed by the attraction between a partial positive charge on the H atom of the amino group and the partial negative charge on the O atom of the peptide bond • Alpha helix - the bonds are formed between repeating atoms on the same polypeptide chain eg collagen • Beta sheets – the bonds are formed between polypeptide chains lying side by side eg. Silk fibrion Individually weak, but strong when repeated R aa aa Δ+ C C ΔN Δ-O H HΔ+ Δ+ ΔH O H N Δ- C R C Δ+ aa aa Alpha-helix Beta pleated sheet Secondary structure Beta sheet Alpha helix Results from interactions between atoms in the polypeptide backbone Tertiary structure – Is the overall three-dimensional shape of a polypeptide ; final shape of a polypeptide – Results from interactions between the side chains of the amino acids Hydrogen bond CH2 CH 2 O H O H 3C CH CH3 H 3C CH3 CH Hydrophobic interactions and van der Waals interactions HO C CH2 CH2 S S CH2 Disulfide bridge O CH2 NH3+ -O C CH2 Ionic bond Polypeptide backbone Tertiary structure • Hydrophobic interactions: Non-polar “R” groups are repelled by water They tend to cluster together Force themselves into the core of a protein Stabilize the overall structure Tertiary structure • Van der Waals interactions: occur between hydrophobic non-polar side chains in close contact Valine H 3C CH CH3 H 3C CH3 CH Hydrophobic interactions and van der Waals interactions Polypeptide backbone Tertiary Structure • Hydrogen bonds – between polar side chains • Examples of amino acid side chains that may hydrogen bond to each other: Two alcohols: ser, thr, and tyr. Alcohol (OH) and an acid : asp and tyr Two acids (COO-): asp and glu Alcohol and amine (NH3+): ser and lys Alcohol and amide (NH2): ser and asn Tertiary structure • Ionic bonds /Salt bridges form between positively and negatively charged side groups • result from the neutralization of an acid and amine on side chains. • The final interaction is ionic between the positive ammonium group and the negative acid group. • Any combination of the various acidic or amine amino acid side chains will have this effect. Tertiary structure • Disulphide bridges form between 2 cysteine residues • Disulfide bonds are formed by oxidation of the sulfhydryl groups (-SH) on cysteine. • Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. • Eg. Insulin contains important disulphide bridges. Disulfide bonds keratin Quaternary Structure – Is the overall protein structure that results from the aggregation of two or more polypeptide subunits – A variety of bonding interactions including hydrogen bonding, salt bridges, and disulfide bonds hold the various chains into a particular geometry. – There are two major categories of proteins with quaternary structure - fibrous and globular. Quaternary structure Fibrous Trimer of alpha-helical Polypeptide chains Globular Globular protein4 Polypeptide chains/ subunits Primarily alpha-helical β Chains Iron Heme α Chains Collagen Structure allows for great strength Rigid resistant to stretch Function = connective tissue in skin, bone, tendons, ligaments. 40% of all human protein Hemoglobin 4 polypeptides bind together to form a round globular shape Functions = carries oxygen Collagen Hemoglobin Quaternary structure Fibrous Globular ribonuclease Beta sheet structure of silk fibroin allows for strength and flexibility Globular proteins are usually round in shape and tend to be a mix of alpha-helical and Beta sheet Determining Protein Structure • X-‐ray crystallography – Is used to determine a protein’s three-‐ X-ray dimensional structure diffraction pattern Photographic film Diffracted X-rays X-ray X-ray beam source Crystal Nucleic acid Protein (a) X-ray diffraction pattern (b) 3D computer model Protein structure summary… • The conformation of a protein determines its function • Proteins are made of polypeptides which are polymers of amino acids • Amino acid polymers are linked by peptide bonds • The amino acid sequence determines the 3-D shape of the Protein • There are four levels of protein structure Primary Secondary Ter@ary Quaternary