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BIOLOGY 2B03 (244)
Kim Dej (39)
Lecture 2

Protein Structure and Function Lecture 2 Summary.docx

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McMaster University
Kim Dej

Protein Structure and Function Lecture 2 Correcting mis-folding: Chaperones Ex. plaque-> in diseases like Alzheimer’s 1. Most protein molecules fold rapidly into their correct configuration 2. Incompletely folded proteins are assisted in refolding by chaperone proteins 3. Misfolded proteins are recognized for degradation Two major types of Chaperones  Prevent improper interaction with other molecules and increase the efficiency of protein folding 1. Molecular Chaperones  Selectively bind to hydrophobic amino acids that are exposed in the native non-conformation, allowing it to fold correctly by preventing the developing polypeptide from associating w/ other proteins or folding prematurely Hsp70 family (Heat Shock Proteins)  Hsp 70 in cytosol and mitochondria  BiP in endoplasmic reticulum  DnaK in bacteria  First discovered b/c they're highly prevalent under stress or increased heat (proteins denature and unfold at high temps) 1. Hsp70 (+ ATP) binds to hydrophobic patch of protein
 a. Terminal phosphate on ATP cleaved, providing energy for many reactions in cell 2. Hydrolysis of ATP to ADP 3. Conformational change in Hsp70, allowing protein to partially fold a. Hsp gets protein folded partially and protein spontaneously completes folding 4. ATP rebinds to Hsp70, allowing Hsp70 to dissociate from fully folded protein a. Hsp can be re-used, goes to another protein after releasing 5. Often release simply allows protein to fold spontaneously 2. Chaperonins  Large cylindrical macromolecule assemblies composed of many proteins o Ex. TCiP in cytosol, GroEL in bacteria/chloroplast, Hsp 60 in mitochondria  Form isolation chamber for newly synthesized polypeptides that allow them to fold w/o interference from other macromolecules  Assist in folding of up to 15% of proteins (mammals) 1. Misfolded protein is initially captured by hydrophobic interactions with rim of “tight” barrel (GroEL) 2. ATP and cap (GroES) bind 3. Conformational change, space enlarges, releasing polypeptide into space and enclosing protein
  GroEL shifts to “relaxed” conformation w/ addition GroES cap, releasing protein within chamber 4. ATP hydrolyses to ADP, this ejects folded protein Hsp-60 Protein Top View  7 Subunits make up each GroEL complex (in Subunit of GroEL eukaryotes (varies), 8 in bacteria)  Intermediate domain acts a hinge allowing each subunit to move relative to each other  1 ATP per subunit Example of change in protein conformation of 1 Hsp60 subunit of GroEL (14 in total)  Chaperonin bound to ATP containing the GroES cap on top on RIGHT  Causes conformational change in each subunit making space for protein to fold Summary  Proteins fold spontaneously, or with help of single molecular chaperone (Hsp70) or with chaperonin complex like GroEl GroEs complex Protein Degradation  Intracellular proteins are selected and marked for degradation by a protein- degrading cellular machine= proteasome OR  Cells must degrade: o Misfolded proteins o Denatured proteins o Proteins in too high concentration’s o Proteins taken up into the cell via endocytosis o Regulate levels of some proteins 2 Step Process 1. Ubiquitinylation  Tagging of the protein by attachment of ubiquitin molecules o Ubiquitin-small protein (76 residues), called ubiquitous b/c it is found in every cell of every species that has been studied  3 enzyme system o E1: Ubiquitin Activating Enzyme: recognizes free ubiquitin in cytosol and picks it up o E2: Ubiquitin Conjugating Enzyme: facilitates attachment of ubiquitin to another molecule o E3: Ubiquitin Ligase: large family of proteins, each member recognizes a different signal on target protein for degradation, interacts w/ E2 to allow conjugation to occur  1. Ubiquitin activated by linkage to E1 2. Activated ubiquitin is transferred to Cys on E2 3. E2 complexes with E3 4. E3 recognizes substrate and transfers ubiquitin to lysine side chain of target substrate 5. Protein is poly-ubiquitinylated - 1 ubiquitin isn't sufficient; ubiquitinylation is a process that occurs in some proteins to assist in folding, poly- ubiquitinylation allows for targeting of protein for degradation 2. Degradation Proteasome= protein degradation machine  Central hollow cylinder formed from multiple protein subunits that assemble as a cylindrical stack  Core has proteolytic activity  Cap on each end form narrow opening through which unfolded polypeptides are threaded  Polypeptide enters through cap where it’s unfolded and threaded in its primary linear structure through the core where it is cleaved  Broken into small peptide (2-24 aa) which are released and further degraded by cytosolic proteins Summary  Ubiquitin carrier enzyme (E2) picks up ubiquitin  Ubiquitin ligase (e3) attaches to target protein  Ubiquitin carrier enzyme (e2) attaches to e3, dropping off ubiquitin before detaching  Poly-ubiquitination occurs through ubiquitin ligase, ubiquitin tag recognized by cap of proteasome  Protein unfolds, threads through core, small peptides extruded out other end Mutant ataxin-1  Found in neurodegenerative disorders-misfolded protein brought to cap of proteasome but it is stuck and has a defensive mechanism that prevents it from unfolding, thus, it builds up in cell creating aggregates/ plaques that are detrimental to cell survival  Problems o 1. Misfolded ataxin protein that isn't being removed from cell (detrimental plaques) o 2. No normal ataxin being produced-harmful to function of cell o 3. Mutant ataxin proteins building up on proteasome preventing normal function and folding of proteins that should be folding Which Proteins are Ubiquinated?  Native cytosolic proteins w/ tightly controlled life spans, e.g. some cell cycle proteins  Proteins that misfold during synthesis in the ER o Both types contain particular aa sequences (hydrophobic) recognized by the E2/E3 ubiquitin enzyme complex  Proteasome-Independent Functions of Ubiquitin in Endocytosis and Signaling o Often there are protein folding events dependent on addition of single ubiquitin molecule o Ex. if protein is involved in signaling, endocytosed to surface of membrane or exocytosed out of cell if it’s a signal Structure-Function Relationships  Some proteins are designed to bind to every type of molecule from simple ions to large complex nucleic acids  Function of almost all proteins depends on their ability to interact/bind other molecules= ligands and substrates (molecules w/ which protein commonly interacts) o Ex. antibodies attach to antigens (target molecule that might be proteins on surface of virus)(at epitope)- require high specificity so shape of protein recognizes shape of ligand o Enzymes bind substrates o Receptors bind signal molecules o DNA binding proteins (recognize double helix shape of DNA and bind to it) Protein-Ligand Interactions Ligand binding must show 1) Specificity  Ability of protein to preferentially bind to 1 unique molecule/ligand or a small number of closely-related molecules 2) High affinity  Strength of binding between protein and ligand (how long they stay attached, not physical strength Both are dependent on the Molecular Complementarity between the ligand and surface of the ligand-binding site Molecular Complementarity  Noncovalent bonds mediate interactions between macromolecules  AD has more non-covalent bonds and a better fit (correlation) o B/C they fit together, more chances for non-covalent bonds to form I. Shape and fit are important:  Non-covalent bonds that mediate the interaction are weak and are only effective when interacting molecules are very close together 0 0 0 0  Protein Structure (1 ,2 ,3 ,4 ) II. The accumulated effect of many interactions  The effect is additive making for a strong association  How many non-covalent interactions? o Varies, some cases may want weak interactions so molecules come apart easily; other cases want strong interactions, etc.  Molecular complementary permits specific binding between proteins  Less Stable: fewer interactions, molecules further apart, incorrect interactions (+ and +)  All proteins bind other molecules, protein-ligand interactions depend on molecular complementarity   Upon folding, aa residues come together to form a nucleotide pocket w/ a strong interaction, cAMP has complementary shape to attach w/ high affinity and strength Binding Affinity  The free energy of interaction between a protein (P) and its ligand (L) can vary greatly  Some affinities for ligands can be so high that dissociation never occurs  Binding affinity is measured by the association constant for the binding equilibrium (K eq  High K eqHigh affinity  Reaction focuses towards the right- more LP complexes in cytosol than individual L and P  K eq a direct measure of affinity o K =eqLP]/ [L][P]  More commonly binding reactions are described in terms of the Dissociation Constant K d o K = [L][P]/[LP] d o Low K =Hdgh affinity Summary  Function of most proteins depends on ability to bind to ligands  Affinity of protein for ligand refers to strength of ligand binding  Specificity refers to preferential binding of 1 or a few related ligands  Ligand-binding sites on proteins and the ligands show molecular complementarity that is dependent upon shape/fit and accumulated non- covalent interactions Enzymes and the Chemical Work of the Cell 6 12  Enzymes allow reactions to occur at a much higher rate (by 10 -10 )by reducing free energy of transition state (catalysis) so reactants can more easily transfer to products  Decrease in free energy suggests a favorable reaction  Rate limiting part: assembly of reactants, affinity between protein (enzyme) and substrate  Catalysis- Enzymes bind their ligands (called substrates) and promote a chemical reaction between them  Enzymes recognizes substrate and brings them closer together so rxn can occur  Often just bringing them together due to the high affinity of each substrate w/ enzyme is sufficient for a reaction, other cases enzyme facilitates reaction between substrates  Enzymes require high specificity and high catalytic power o Determined by certain amino acid side chains within enzyme’s active site Two functional regions of active site 1. Binding site/pocket- determines specificity 2. Catalytic site-promotes reaction Binding  Cleaves substrate into 2 products, or if we bring 2 substrates together may be sufficient to catalyze reaction Catalysis Enzyme Kinetics  The catalytic activity of an enzyme described by max and K m o V max= maximal velocity of reaction at saturating substrate Release concentrations (limited by # of enzymes present) o Eventually adding substrates won’t increase rate of reaction- b/c enzyme limited by # of substrate binding/active sites present (10 enzymes can only bind to 10 active sites at a time  Km(Michaelis constant)= concentration of substrate at which reaction velocity is half maximal (measure of affinity of an enzyme for the substrate)  High affinity substrate= low m , Low affinity substrate= high m o Vmax remains the same for both (same # of active sites)  At low affinity we need more substrate at half Vmax to increase
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