Topic 9 - Protein Structure PDF

🥓 Topic 9 - Protein Structure Key features of Proteins General Structure of an Amino Acid Amino acids consist of a central carbon atom (the α carbon) covalently bonded t...

🥓 Topic 9 - Protein Structure Key features of Proteins General Structure of an Amino Acid Amino acids consist of a central carbon atom (the α carbon) covalently bonded to: an amino group (-NH2) a carboxyl group (-COOH) a hydrogen atom (-H) a distinctive R group (side chain) Define Amino Acid Residue An amino acid residue is what remains of an amino acid after it has been joined by a peptide bond to form a protein Topic 9 - Protein Structure 1  Zwitterion Form of Amino Acids* Amino acids have –NH2 group and –COOH group and both groups undergo ionisation in water acquire + and – chargers low pH - protonated - gain of H+ high pH - deprotonated - loss of H+ Zwitterion - both positive & negative charge (Isoelectric point - amino acids have an overall charge of zero) Overall neutral at physiological pH (7.4) Which structure of an amino acids decides its classification chemical + physical properties of the R groups Topic 9 - Protein Structure 2  what are “pK values” A measure of the strength of an acid on a logarithmic scale if the solution pH < pK value of amino acid the R group will be protonated pH - 7.4, pK - 10.5 if the solution pH > pK value of amino acid the R group will be deprotonated pH - 7.4, pK - 2.8 Handerson-Hasselbalch Equation At the isoelectric point, the + and – charges are equal and concentration of acid is equal to base. pKa = acid dissociation constant Topic 9 - Protein Structure 3  Peptide Bond Formation Peptide bonds are planar, Why are C-N unable to rotate ? peptide bond C-N has partial double bond characteristics Unable to rotate – contributes to planarity Out of Cis/Trans in Peptide Bonds, why is the Trans form favoured Topic 9 - Protein Structure 4  Trans form is strongly favoured because of steric clashes (unnatural overlap of any two non-bonding atoms in a protein structure.) Define the term ‘Isoelectric Point’ of Proteins The isoelectric point, pI, of a protein is the pH at which there is no overall net charge BASIC PROTEINS pI >7 Contain many positively charged (basic) amino acids ACIDIC PROTEINS pI < 7 Contain many negatively charged (acidic) amino acids If pH < pI protein is protonated If pH > pI protein is deprotonated Define Oligopeptides A few amino acids in length Conjugated proteins* A protein with just amino acids as a building block is a simple protein; those that contain additional units, such as a nucleic acid, a lipid or a metal etc., are called conjugated proteins. Topic 9 - Protein Structure 5  Primary Structure Primary structure: the linear amino acid sequence of the polypeptide chain Secondary Structure Definition local spatial arrangement of polypeptide backbone. α helix, β–pleated sheet Secondary Structure - The a-helix 3.6 amino acids per turn Right Handed Helix the backbone C=O group of one residue is H-bonded to the –NH group of the residue four amino acids away. Topic 9 - Protein Structure 6  Sequences that affects a-helix stability Small hydrophobic residues → strong helix formers. Pro —> helix breaker because the rotation around the N-Ca bond is impossible. Gly —> helix breaker because the tiny R-Group supports other conformations. Secondary Structure - B-strand Tertiary Structure Quaternary Structure Lecture 2 Topic 9 - Protein Structure 7  Protein misfolding A nascent protein can either form into properly folded protein - normal function toxic clump protein - non functional Takes place during CO-TRANSLATIONAL + POST TRANSLATIONAL Fibrous Proteins (Role, Shape & Example) ROLE = support, shape + protection more rigid, less flexible than globular proteins single type of repeating secondary structure SHAPE = long strands and sheets example = collagen Globular Protein (Role, Shape & Example) ROLE = Catalysis + Regulation Compact Shape e.g. Carbonic Anhydrase water soluble Topic 9 - Protein Structure 8  Collagen Collagen: Main fibrous protein in animals, provides structural support. Found in: Skin, tendons, ligaments, cartilage, bone, teeth, membranes, and blood vessels. Stiffens with Calcium Why does Collagen have a Glycine a.a. at every 3rd position of the a-helix chain Glycine's non-polar nature and repetitive sequence create tight, compact structures keeps collagen in a fibrous shape How are collagen fibrils formed Composition: Three preprocollagen chains —> form a triple helix. Stability: Stabilized by direct inter-chain hydrogen bonds and water-mediated hydrogen bonds. Formation Process: Procollagen → transformed into tropocollagen. Topic 9 - Protein Structure 9  Tropocollagen Linking: Tropocollagen molecules are covalently linked via aldol cross-links to create collagen fibrils. Name 2 types of Globular Tertiary Structures Motifs Domains Motifs (A type of Globular Tertiary Structure) Small structures. Folding includes secondary structure elements like alpha helix and beta sheet. Can exist as loops and barrel-like shapes. Domains Larger structures. Part of a Polypeptide chains fold into specific shapes with specific functional roles. Topic 9 - Protein Structure 10  Folding of Water Soluble Proteins polypeptide chains fold so that hydrophobic side chains are buried within the protein polar, charged chains are on the surface Folding of membrane proteins Membrane proteins enable the movement of molecules in and out of cells through a pore in the protein. These proteins have an inside-out design inside of the pore having hydrophilic polar side chains, while the outside has hydrophobic amino acids. This setup permits the transfer of water-soluble molecules into the cell. Topic 9 - Protein Structure 11  Heteromers and Homomers (Quaternary) Heteromers: Protein complexes formed by different types of polypeptide chains. Homomers: Protein complexes formed by repeating/multiple copies of a single polypeptide chain. Honomers (regulation, efficiency & error control, stability) Homomers: Protein complexes formed by repeating/multiple copies of a single polypeptide chain Efficiency and Error Control: Using the same chain repeatedly is genetically efficient and aids in minimizing errors. Stability: Homomers are more stable because they bury a larger portion of their surface area. DNA Binding of P53 p53 binding to DNA increases its activity. Low DNA binding activity allows cells to survive. High binding affinity leads to cell death (apoptosis). Mutations in p53 —> increase DNA binding cooperativity —> excessive transcription —> cancer. p53 Tetrameric Complex (DNA Binding of P53) p53 forms a tetrameric complex with four subunits when binding to DNA Topic 9 - Protein Structure 12  Tetrameric transcription is advantageous because if one subunit has an error, the complex's binding affinity may decrease, but some functionality can still be maintained. Monomeric Transcription Factor (DNA Binding of P53) a single mutation in the DNA-Binding domain or in the DNA response element (RE) can abolish binding Protein Misfolding (how are they marked and what do chaperons do) Misfolded proteins are typically: Marked with ubiquitin and directed to the ubiquitin-proteasome system for degradation (the normal pathway). Given a second chance to fold correctly. Chaperones assist in protein folding by helping them adopt the right shape, requiring the use of ATP. Neurofibrillary tangles in Alzheimer’s disease (Tau Protein Aggregation) Tau protein aggregates inside neurons. These aggregates lead to the depolymerization of microtubules and disrupt their normal function. Aggregated tau proteins themselves also contribute to neuronal dysfunction. Topic 9 - Protein Structure 13  Describe the process of Polyacrylamide Gel Electrophoresis Create a gel by polyacrylamide. Mix samples with a loading buffer. Load samples into the wells. Submerge the gel in an electrophoresis chamber. Apply an electric field to separate molecules. Molecules separate based on size and charge. Smaller molecules move faster + migrate farther Turn off the power remove the gel from the chamber. Stain the gel to make bands visible. Compare band positions to molecular weight markers for size and quantity information. Topic 9 - Protein Structure 14  What is SDS (Sodium Dodecyl Sulphate) - 1D SDS Page anionic detergent that unfolds proteins and provides them with extra negative charges SDS Page separates proteins based on molecular weight Sample Prep: Proteins are treated with SDS to give them a negative charge and denature them. Gel Formation: Create a polyacrylamide gel Sample Loading: Load prepared protein samples into the gel and seperate depending on weight Carry out Electrophoresis two dimensional SDS Page 2 Dimension (SDS-PAGE): First Dimension (Isoelectric Focusing - IEF): Proteins loaded onto a pH gradient strip. Electric field separates proteins to their isoelectric point (pI). Separation based on charge. Second Dimension (SDS-PAGE): The first-dimension strip is placed on a polyacrylamide gel. Proteins separate based on molecular weight. An electric field is applied again to create spots based on size/weight. Topic 9 - Protein Structure 15  Visualization and Analysis: Stain the gel to reveal protein spots. Result is a 2D map of proteins with spots showing pI and molecular weight. Used for protein identification and quantification in complex mixtures Topic 9 - Protein Structure 16