Learning objectives At the end of this lecture a student should be able to :• Classify the amino acids based on polarity, nutritional requirements, side chains and chemical side chains • Describe the pathways of Protein turnover. Explain Ubiquitin proteosome pathway • State the four levels of protein structure and explain how the sequence of amino acids leads to the final three dimensional configuration of the protein • Explain Protein misfolding and its clinical implications • Analyse the Concept of zwitter ions and its clinical applications L-Form Amino Acid Structure Carboxylic group Amino group + H3 N R group COO a - H H = Glycine CH3 = Alanine Compounds having same structural formula , but differ in spatial configuration are known as stereoisomerism. Penultimate Carbon Mirror Images of Amino Acid a Mirror image Enantiomers a Amino Acids Classification • • • • Based on polarity Based on side chain Based on metabolic function Based on nutritional requirement Based on Side chain Classes Side chain Polarity Example I Aliphatic Apolar Gly, Ala, Val, Leu, and Ileu. II Sulphur Apolar Cys* and Met III Aromatic Apolar Phe, Tyr & Trp IV Hydroxyl Polar Ser, Thr and (Tyr) V Acidic Polar Asp , Asn and Glu, Gln VI Basic Polar Lys , Arg and His VII Imino Apolar Pro USMLE ! Aliphatic side chains (non-polar) Imino acid ( Non-polar) • 1. 2. 3. Glycine : Smallest amino acid Inhibitory neurotransmitter Most common amino acid of collagen • Valine, Leucine and isoleucine : 1. Leucine – ketogenic amino acid 2. Maple syrup urine disease • Proline – imino acid , disrupts amino acid structure Aromatic amino acids Indole ring • 1. 2. Phenyl Alanine : Essential amino acid : forms tyrosine Def : phenyl ketonuria ( phenyl alanine hydroxylase deficiency) • 1. 2. 3. 4. Tyrosine : Precursor of melanin Catecholamines – dopamine, norepinephrine and epinephrine which act as neurotransmitters Parkinson disease : def of dopamine synthesis of thyroxine . • 1. 2. 3. Tryptophan : (indole ring) Melatonin - circadian rhytm Serotonin – mood alterations Source of Niacin – def causes hartnup’s disease Sulphur containing amino acids • Cysteine : 1. Used to make disulfide bonds – secondary structure of proteins 2. Glutathione – detoxifying agent and free radical scavenger. • • • • Pharmacology : N –acetyl cysteine Paracetamol toxicity Cystic fibrosis Cyclophosphamide toxicity – treatment • Methionine : 1. S-adenosyl Methionine : methyl donor 2. Homocysteine – derived from methionine Side chains with Basic groups (polar ) Guanidino group Imidazole group • Lysine and arginine – due to positive charges , main components of histone proteins. • 1. 2. 3. Arginine : Synthesis of nitric oxide Release of urea Semi-essential amino acid – positive nitrogen balance • Histidine : 1. Histamine – major inflammatory mediator . Acidic groups and their amides (polar) Side chains with –OH groups 21st and 22nd aminoacid • Selenocysteine • Pyrrolysine Based on Nutritional requirement Essential Amino Acids • Not synthesized in the body • Essential in diet Non - Essential Amino Acids • Synthesized in the body Nutritionally essential mnemonic – MATTVILPHLY OR PVT TIM HaLL USMLE ! Classification based on essentiality PVT TIM HaLL Arginine also. Based on Metabolic function Classification of Amino Acids by Polarity POLAR Acidic Asp NONPOLAR Glu Ala Val Neutral Asn Cys * Gln Ser Tyr Thr Basic Arg His Lys Ile Gly Phe Trp Leu Met Pro Non-polar substances likely to be inside the cell membrane , polar things OUTSIDE ! •Cysteine is polar because of its thiol group but free cysteine residues are found to associate with hydrophobic regions of proteins USMLE ! Zwitter ion • An amino acid acts as a zwitterion, i.e., it can be either: 1) Positively charged in an acid solution 2) Negatively charged in an alkaline solution 3) Neutral at the isoelectric point. Formation of Peptide Bond Formation of Peptide Bonds by Dehydration Amino acids are connected head to tail NH2 1 COOH 2 NH2 COOH Dehydration Carbodiimide -H2O O NH2 1 C N H 2 COOH Amino acids are joined by peptide bonds • Characteristics of PEPTIDE BOND – Partial double bond(The distance is 1.32 A which is midway b/w single bond (1.49 A)and double bond(1.27A)) – Rigid and Planar – The partial double bond renders the amide group planar, occurring in either the cis or trans isomers. – In the unfolded state of proteins, the peptide groups are free to isomerize and adopt both isomers; however, in the folded state, only a single isomer is adopted at each position (with rare exceptions). Numbering of amino acids in Peptides and Proteins • Free alpha amino group at one end – AMINO TERMINAL( N –TERMINAL ) • The amino acid contributing the alpha amino group is called as first amino acid. • Biosynthesis of protein starts from this terminal end only. • Free carboxy group on other end – CARBOXY TERMINAL(C –TERMNAL ) –> LAST AMINO ACID Amino acid sequences • Greek Proteios meaning PRIMARY • BUILDING BLOCKS OF THE BODY • 3/4TH DRY BODY WEIGHT = PROTEINS • CONTAIN C, H , O, N as major components, while S and P are minor constituents. • N content of ordinary proteins is 16% by weight. PEPTIDES • Proteins are made by polymerisation of amino acids through peptide bonds. • 2 AA joined to form a DIPEPTIDE • 3 AA -> TRIPEPTIDE • 4 AA-> TERTAPEPTIDE • 5- 10 AA -> OLIGOPEPTIDE • 10- 50 POLYPEPTIDE • >50 PROTEIN 3 AA means 20 3 COMBINATIONS = 8000 possible types of peptides. An ordinary protein with 100 amino acid = 20100 possible combinations !!! Primary Assembly Secondary Folding Tertiary Packing Quaternary Interaction PROCESS STRUCTURE Biology/Chemistry of Protein Structure PRIMARY STRUCTURE OF PROTEINS The sequence of the amino acids in a polypeptide chain denotes its primary structure. • • • • • linear ordered 1 dimensional sequence of amino acid polymer by convention, written from amino end to carboxyl end • a perfectly linear amino acid polymer is neither functional nor energetically favorable folding! primary structure of human insulin CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT Secondary structure • The term secondary structure denotes the configurational relationships between residues which are about 3-4 amino acids apart in linear structure. • It is stabilized by non-covalent bonding Secondary Structure • non-linear • 3 dimensional • localized to regions of an amino acid chain • formed and stabilized by hydrogen bonding, electrostatic and van der Waals interactions (Pauley and Corey 1951) • Spiral structure • Polypeptides form the back bone and the side chains of amino acids extend b/w NH & C=O groups • Distance b/w adjacent a.a is 1.5 A • 3.6 aa per turn • RIGHT HANDED • IT IS THE MOST COMMON AND STABLE CONFORMATION OF S POLYPEPTIDE CHAIN • Abundant in hemoglobin and myoglobin and and absent in chymotrypsin. Proline and hydroxy proline will not allow the formation of alpha –helix. The spirals look like they are going down clockwise. H N C C O H N R C C O H C C N O C H C O N C O C H C C N O C O H N R R R R R R H N R R R R R R R Secondary structure a -helix (Pauling and Cory 1951) • the polypeptide in beta-pleated sheet is almost fully extended. • The distance b/w adjacent a.a. is 3.5 A. • It is stabilized by hydrogen bonds between NH and C=O groups of neighbouring polypeptide segments. • Adjacent strands can run in same direction(parallel) or opposite direction(anti-parallel) with regard to amino and carboxy terminal. • Abundant in Silk (anti-parallel), Flavodoxin (parallel), Carbonic – anhydrase (both). Secondary structure beta pleated sheet O C C H C N C O N H O C N H C O C H C N C C N H C C O N H O H N C O C C O H C N C O N H O H N C O C C C N H C C H N C O C O C H C N N H C O O H N C N H C C O C Protein Folding • Occurs in the cytosol • Involves localized spatial interaction among primary • ADVANTAGES :structure elements, i.e. the amino acids Tumbles towards conformations that • May or may not involve reduce E (this process is chaperone proteins thermo-dynamically favorable) Yields higher structures of proteins. Active sites open up!!! • Non-linear Tertiary Structure • 3 DIMENSIONAL STRUCTURE OF A PROTEIN • Global but restricted to the amino acid polymer • Formed and stabilized by hydrogen bonding, non-covalent interactions like hydrophobic packing toward core and hydrophilic exposure to solvents. • A globular amino acid polymer folded and compacted is somewhat functional (catalytic) and energetically favorable interaction! • It represents the three 3D structure of the protein. • It defines the steric relationships of a.a which are far apart from each other in the linear sequence , but are opposed each other by non-covalent interactions. • Thermodynamically , the most stable structure . • DOMAIN :- It is a term used to denote a compact globular functional unit of a protein.A domain is a relatively independent region of the protein , and may represent a functional unit. Quaternary Structure of a Protein • Certain polypeptides aggregate to form one functional unit. This is called as Quaternary Structure . • non-linear • 3 dimensional • global, and across distinct amino acid polymers • formed by hydrogen bonding, covalent bonding, hydrophobic packing and hydrophilic exposure • favorable, functional structures occur frequently and have been categorized . Structure of Hemoglobin • Stabilized by H-bonding, electrostatic bonds , hydrophobic interactions and Van Der Walls forces. • Each polypeptide chain is termed as a subunit or monomer. • Eg. Hemoglobin :- 2 α, 2 β chains – Creatine Kinase is a dimer – LDH is a tetramer – Aaspartate transcarbamoylase has 6 subunits. • ENZYMES :– Enzyme catalysis needs precise binding of the substrate to the active site of the enzymes. – This depends on structural conformation of the active sides so that it is precisely oriented for substrate binding. • TRANSPORT PROTEINS – Hb has a quaternary str with 2 α and 2β subunits.Binding of O2 to one heme subunit facilitates oxygen binding by other subunits. – Binding of H+ and CO2 promotes release of O2 from hemoglobin. This is called as BOHR effect. • In Primary structure any amino acid change /deletion/ replacement of any one acid by another can cause abnormalities in its function. – Eg. Sickle cell anaemia :- In 6nd position of β-chain of Hemoglobin if there is Valine instead of Glutamic acid . Classification based on Functions :1. Catalytic proteins e.g.enzymes 2. Structural proteins e.g. collagen , elastin, keratin 3. Contractile proteins e.g. myosin, actin, flagellar proteins 4. Transport proteins , e.g. haemoglobin, albumin 5. Regulatory proteins e.g. ACTH , insulin , GH 6.Genetic proteins , e.g. Histones 7. Protective proteins e.g. Immunoglobulins • Classification based on Nutritive Value 1)Nutritionally rich proteins or Complete proteins or First class proteins: They contain all essential a.a. in required proportion for ideal body growth and development. E.g egg albumin , milk casein • 2.Incomplete proteins :- They lack one essential amino acid. They cannot sustain body growth in young individuals even if they are able to maintain growth in adults. Eg Pulses are deficient in methionine and cereals are deficient in lysine .If both are combined in diet , good growth can be obtained. • 3.Poor proteins :-They lack in many essential a.a. and a diet based on them cannot sustain the original body weight e.g. Zein of corn lacks Trp and Lys. • Classification based on shape . 1.Globular proteins :-They are spherical or oval in shape.(l/b <10) eg albumin, globulin 2.Fibrous proteins :- They are elongated or needle shaped and resist digestion .e.g. collagen, elastin, keratin etc. NITROGEN BALANCE Nitrogen balance = Nitrogen ingested - Nitrogen excreted (primarily as protein) (primarily as urea) Nitrogen balance = 0 (nitrogen equilibrium) protein synthesis = protein degradation Positive nitrogen balance protein synthesis > protein degradation Negative nitrogen balance protein synthesis < protein degradation Negative nitrogen balance • • • • • • Starvation PEM Uncontrolled Diabetes Mellites Def of any essential aminoacid Infection Surgery, Burns MCQ1)A mixture of ala,arg,his,gly,glu is subjected to electrophoresis at pH 7. -ve • Identify this amino – A.Glycine – B. Arginine – C.Glutamate – D.Valine – E.ALANINE +ve MCQ2 • Several complexes in the mitochondrial ETC contain non-heme iron tightly bound to a thiol group of which amino acid? – A. Glutamine – B.Methionine – C.Tyrosine – D.Cysteine – E.Serine • Thank you