Protein Structure and Function Chapter 4 PDF

Protein Structure and Function Chapter 4 1 Chapter Contents THE SHAPE AND STRUCTURE OF PROTEINS HOW PROTEINS WORK HOW PROTEINS ARE CONTROLLED (HOW PROTEINS ARE STUDIED) – Not covered in class 2 Pr...

Protein Structure and Function Chapter 4 1 Chapter Contents THE SHAPE AND STRUCTURE OF PROTEINS HOW PROTEINS WORK HOW PROTEINS ARE CONTROLLED (HOW PROTEINS ARE STUDIED) – Not covered in class 2 Protein Structures and Functions Proteins are the main building blocks from which cells are assembled. ~55% of dry cell weight is protein! Proteins have a multitude of functions due to their ability to adopt many different shapes. Rule: Shape determines function! 3 THE SHAPE AND STRUCTURE OF PROTEINS 4 The Shape of a Protein Is Specified by Its Amino Acid Sequence Proteins are assembled from a set of 20 different amino acids and linked together by a peptide bond. Each protein has a unique order of amino acid and therefore a unique structure and function. What are the properties of the 4 R- Polypeptides have flexibility groups in this image? Many single bonds can rotate Allows protein to fold, in principle, in different ways. Peptide bond has a partial double bond character due to the resonance of the amides. Rotation around this bond is restricted. 5 Lends stability to peptide bond. Proteins Fold into a Conformation of Lowest Energy Many macromolecules including proteins can self-assemble (fold) to form more complex structures due to interactions (i.e. hydrogen bonds, ionic bonds, …) within the molecule Folding is energetically favorable process Peptide bond, on the other hand, is enzyme driven not self-assembled! The most stable three-dimensional structure of a particular polypeptide is called the native conformation – “Energy Muntau, Ania & Leandro, João & Staudigl, Michael & Mayer, Felix & minimum of the system” Gersting, Søren W. (2014). Innovative strategies to treat protein misfolding in inborn errors of metabolism: pharmacological chaperones and proteostasis regulators. Journal of inherited metabolic disease. 37. 10.1007/s10545-014-9701-z. 6 Protein Folding and Stability 1 Noncovalent bonds and interactions are individually weaker than covalent bonds but collectively can strongly influence protein structure and stability Hydrophobic forces help proteins fold into compact conformations. Drives the movement of hydrophobic R-groups into a tightly packed hydrophobic core away from water. 7 Protein Folding and Stability Mutations in the nucleic acid sequence of a gene can sometimes cause substitutions of one amino acid for another in the encoded protein and severely disrupt the normal structure of a protein. Example Substituting an amino acid with a hydrophobic/non- polar R-group with one that is charged/acidic/basic Methionine to arginine 8  Chaperone Proteins Can Guide The Folding Molecular chaperones make the protein folding process more efficient and reliable. Defective folding and/or impaired chaperones have been implicated in a variety of diseases such as neurodegeneration, heart disease, and cancer. Molecular chaperone are proteins that can bind to newly synthesized or partially folded chains and help them fold along the most energetically favourable pathway. act as isolation chambers that help a polypeptide fold. Requires ATP hydrolysis 9 Proteins Come in a Wide Variety of Complicated Shapes Although the three-dimensional structure of each protein is unique, there are some common folding patterns. 10 Protein Structure Depends on Amino Acid Sequence and Interactions The overall shape and structure of a protein are described in terms of four levels of organization Primary structure—amino acid sequence Secondary structure—local folding of polypeptide Tertiary structure—three-dimensional conformation Quaternary structure—interactions between monomeric proteins to form a multimeric unit 11 Primary Structure Primary structure refers to the amino acid sequence held together by peptide bonds. By convention, amino acid sequences are written from the N- terminus to the C-terminus, the direction in which the polypeptide was synthesized COO- Carboxyl end The sequence of amino acids is specified by the order of nucleotides in the corresponding messenger RNA The primary structure is important because the order and identity of amino acids directs the formation of the higher-order (secondary and tertiary) structures Determines the biological activity of a protein Many genetic diseases result from abnormal amino acid sequences. Sickle-cell anemia, phenylketonurea, Tay-Sachs disease,… NH3+ COO- 12 Denaturation and Peptide Bond Peptide bond is quite strong, not easily broken. Not affected by denaturation (pH, reducing agents, salts, heat…). Denatured proteins are more easily digested due to the unfolding of the peptide chains. HCL in stomach. 13  Secondary Structure The secondary structure of a protein forms from hydrogen bonding between N-H and C=O groups along the polypeptide backbone Stabilizes the protein. Result in two major patterns α helix and β sheet Long, rod-like Parallel coils - Less stable Form the Peptide bond than anti- framework for parallel many elongated Rigid structures proteins such as Form the core of keratin many proteins Anti-parallel 14 FYI: β-Sheets Many species of fish, insects, plants and micro-organisms living in cold environments produce antifreeze proteins (AFP) that prevent ice crystals forming. AFP bind to ice crystals to inhibit growth and damage to the cell. Parallel β-helix structure binding to ice. 15 Amino acids vary in their tendencies to form alpha helices or beta sheets. FYI: β-turn Breaks or kinks a helix Often seen as the first residue of a helix, it is presumed due to its structural rigidity. 16 Super Secondary Structures - Motifs Beta-strand Beta-strand Beta-strand Beta-strand Motifs are simple combinations of secondary structural elements (such as alpha helices and and beta pleated sheets) that frequently occur in proteins. Can include “unstructured” regions such as loops which join structured elements together. Examples Four beta strands Beta-alpha-beta Helix-turn-helix very common motif found in many regulatory proteins such as transcription factors. 17  Misfolded Proteins Can Form Amyloid Structures That Cause Disease Folding a protein into ⍺ helices or β sheets depends on amino acid sequences. A mutation in particular stretches of the normal protein sequence can spontaneously initiate misfolding. Can damage cells and tissues Thought to contribute to some neurodegenerative disorders such as Alzheimer’s and prion diseases. Amyloid fibrils 18 Normal brain image Alzheimer’s Creutzfeld-Jakob disease Tertiary Structure The tertiary structure reflects the full three-dimensional conformation formed by an entire polypeptide chain, including interactions of the R-groups Tertiary structure can include non-amino acid-based cofactors. Generally, without its cofactors, a protein is inactive and often unstable. 19 Protein Domains The protein domain is a distinct functional and/or structural unit in a protein. Not considered part of the four levels of organization Usually has a specific function Proteins with similar functions often share a common domain Proteins with multiple functions usually have a separate domain for each function, ATP-binding domain in kinase like modular units from which globular - Adenine region proteins are constructed - Ribose region - Phosphate region 20 Quaternary Structure Term applies specifically to multimeric proteins Can contain multiple copies of the same protein subunit or different polypeptides. Dimer – 2 polypeptide chains Tetramer – 4 polypeptide chains The bonds and forces maintaining quaternary structure are the same as those responsible for tertiary structure The process of subunit formation is usually spontaneous Sometimes, molecular chaperones are required to assist the process 21 Complex Quaternary Structures Protein subunits can assemble into complex quaternary structures. Can produce extended protein filaments Actin filaments or microtubules are major components of the cytoskeleton Many large structures are built from mixtures of one or more types of protein plus RNA or DNA molecules Ribosomes consist of proteins (purple) and rRNA (brown) https://commons.wikimedia.org/w/index.php?curid=2839678 22 Proteins Can Be Classified into Families A protein family is a group of proteins that share a common evolutionary origin, have related functions and similarities in amino acid sequence or structure. FYI: Naming of proteins Suffix –in for proteins Hemoglobin, insulin, secretin,… Suffix –ase for enzymes Lactase, protease, maltase,… Integrin family 23 Protein Categories Myoglobin Proteins can be divided into two broad categories, globular and fibrous. Globular proteins Most proteins, including enzymes, are globular proteins Fold up into compact shape like a ball with irregular surface More easily denatured Fibrous proteins Proteins with a simple, filamentous, elongated shape. Provide structural support for cells and tissues. Hydrophobic and not as easily denatured as globular proteins Example: collagen 24 Extracellular Proteins Are Often Stabilized by Covalent Cross-Linkages Extracellular proteins often require additional covalent bonds to stabilize their structure. Most common covalent cross-links in proteins are disulfide bonds. Formed between two cysteine amino acids. Disulfide bond formation not favored in cytoplasm because reducing agents (Vitamins A, C, E, glutathione,…) would break bond. Disulfide bonds form primarily in the oxidizing environment of the ER. Example Insulin Lysozyme in saliva Keratin in hair Insulin 25 Curly or Straight Hair? Hair consists of many interwoven keratin α- helices extensively cross-linked with cysteine disulfide bonds. Helices are also stabilized by hydrogen and ionic bonds. When hair gets wet, water molecules intrude into the keratin strands, disrupt hydrogen bonds Keratin in hair and the helices slip past each other. If hair is blow dried, new bonds form and the hair can retain a new shape for a short time. A permanent wave uses a reducing agent to disrupt cysteine bonds. Hair is then curled and an oxidizing agent is used to reform the cysteine bonds in the helices. 26 HOW PROTEINS WORK Form and function are inextricably linked! 27 All Proteins Bind to Other Molecules ALL proteins bind to other molecules in a specific manner. Can be weak and short-lived Can be very tight and long-term The binding of a protein to other biological molecules shows specificity Substance bound to protein is called ligand Effective binding requires simultaneous formation of many weak, noncovalent interactions AND surface contours of ligand has to fit surface contours of protein 28 Binding sites allow proteins to interact with specific ligands. The folding of the polypeptide chain typically creates a binding site in such a way that it can form a set of noncovalent bonds only with certain ligands. Amino acids that make up binding site are not necessarily located right next to each other! Changes in even one amino acid within the polypeptide can change tertiary structure and function! 29 Antibodies, an example of a protein that binds to very specific ligands Antibodies are proteins made by B cells as a defense against pathogens. Each resting B cell carries a different membrane-bound antibody on its surface that serves as a receptor for recognizing a specific antigen – target molecule. When an antigen binds to this receptor, the B cell is stimulated to divide and secretes large amounts of the same antibody. 30 Antibody Diversity Each antibody recognizes one very specific antigen and binds only to that antigen. The polypeptide loops in its variable domains determine the specificity an antibody has for its antigen. Humans produce billions of different antibodies to match the billions of potential antigens we might encounter. 31 Antibody Diversity 2 Antibody diversity is produced by the mutation and random recombination of approximately 300 different gene segments encoding the light and heavy chain variable domains in precursor cells that are destined to become B cells. https://pressbooks.bccampus.ca/conceptsofbiologygunness/chapter/23-3-antibodies/ 32 FYI: Autoantibodies Mediated Autoimmune Diseases More than 2.5% of the population is affected by autoantibody-driven autoimmune diseases. Graves’ disease ~3 million Americans Autoantibodies mimic TSH and activate the TSH receptor in an unregulated manner, thereby causing hyperthyroidism Rheumatoid arthritis ~1.3 million U.S. adults Autoantibodies lead to bone and cartilage destruction in joints Myasthenia gravis ~36,000 to 60,000 cases in the United States Autoantibodies bind to acetylcholine receptors and block neuromuscular impulse transmission = extreme muscle weakness/paralysis and potentially death through respiratory arrest in severe cases. Lupus (~1.5 million), multiple sclerosis (Epstein-Barr virus infection; ~1 million), type 1 diabetes (~1.25 million), …. 33 Apo- and Holoenzyme Many enzymes are regulated by molecules that are not part of the enzyme itself Apoenzyme - Inactive enzyme Activation of the enzyme occurs upon binding of an organic or inorganic cofactor. Inactive Active 34  Molecules That Regulate Enzymes Some enzymes require non-protein molecules/ions for catalytic activity, often because they function as electron acceptors 1. Cofactors 1. ​Inorganic ions such as Zn2+, Mg2+, and Fe2+, can reversibly interact with enzymes 2. ​Organic molecules (coenzymes) such as NADH or FADH2, can reversibly interact with enzymes 2. Prosthetic groups are atoms or non-amino acid molecules that are permanently attached to proteins 35 Coenzymes Coenzymes are organic molecules Generally, derivatives of vitamins Vitamin Coenzyme Function Niacin (B3) NAD+ Oxidation or hydrogen transfer Riboflavin (B2) FAD Oxidation or hydrogen transfer Pantothenic Acid (B5) Coenzyme A (CoA) Acetyl group carrier Association with apoenzyme is usually transient 36 Common Coenzymes Coenzyme Q10 Electron carrier required in the electron transport chain and as an antioxidant. Sold as a supplement Evidence that supplementation improves bioenergetics, cardiovascular disease and inflammation. https://doi.org/10.3389/fphys.2018.00044 Coenzyme NAD+/NADH Electron carrier involved in redox reactions such as oxidation of alcohol. Mitochondria – OXPHOS (ATP production) Sold as supplement Potential treatment for chronic fatigue syndrome in conjunction with Q10. https://www.mdpi.com/2072- 6643/13/8/2658/htm Alcohol dehydrogenase Acetaldehyde dehydrogenase 37 Prosthetic groups Organic (i.e. vitamins) or inorganic (i.e. metals) molecules/ions tightly bound to enzyme Example Heme in hemeproteins Heme Required by hemoglobin to carry oxygen catalase and peroxidase to convert H2O2 to H2O and O2. Contains iron which participates in redox reactions. 38 Factors that affect the functioning of enzymes For every enzyme, there are optimal conditions under which it is most effective. Enzymes are affected most by changes in Temperatures Affects movement of the substrate and enzyme pH Affects the enzyme’s shape and reactivity 39 Factors that affect the functioning of enzymes 2 Temperature Increase in temperature will increase collisions between substrate and enzyme = more product produced Too large an increase in temperature = enzyme denatures/active site loses shape = rate of reaction the same as if no enzyme is present Lower temperatures just reduce speed of collisions = less product formed Optimal temperature varies with enzyme. Devil’s Kitchen – Lassen National Park 40 Temperature affinities of different types of bacteria Refrigerator temp Keeping food warm ≤ 4 ℃ (40oF) temp ≥ 63 ℃ (145oF) Ideal growing temperatures for human pathogens Salmonella 35℃ – 37℃ - “stomach flu” Staphylococcus aureus and E. coli 37℃ 41 Factors that affect the functioning of enzymes 3 pH Suppose an enzyme has an optimum pH around 7. At a pH of 7, a substrate attaches itself to the enzyme via two ionic bonds. 42 Factors that affect the functioning of enzymes 4 If the pH is higher or lower than 7, the ability of the enzyme to bind to substrate is affected Extreme pH denatures protein At lower pH, COO- picks up H+ and ionic At higher pH, NH3 will lose H+ and bond with substrate cannot form ionic bond with substrate cannot form 43 Applications to pH and enzyme function Intestine Stomach What might happen if you What would happen if your take too many antacids? stomach emptied its contents into Acid suppression therapy and allergic reactions the small intestine too quickly? Allergo J Int. 2015 Dec; 24(8): 303–311 Bariatric surgery and dumping syndrome 44 Enzyme Kinetics A Closer Look Than In The Book Study of the rate of enzyme-catalyzed chemical reactions 45  Enzymes Are Powerful and Highly Specific Protein Catalysts Are specific for a single type of reaction Bring reactants together in precise orientations Make reactions more likely https://courses.lumenlearning.com/boundless-biology/ Induced Fit: Might require cofactor Both enzyme and substrate undergo dynamic conformational changes upon binding. Convert substrates to products while The enzyme contorts the substrate into its transition remaining unchanged themselves. 46 state, thereby increasing the rate of the reaction. KNOW the common classes of enzymes! 47 Enzyme Kinetics 1 Rate of reaction = How fast the substrate is turned to product (or how quickly the substrate disappears) Reaction rates are influenced by factors such as concentrations of substrates Substrate concentration of enzymes Products Inhibitor concentrations of products Presence of inhibitors Enzyme 48 Enzyme Kinetics 2 Enzyme catalyzed reactions usually show a hyperbolic relationship between the rate of reaction and the concentration of substrate Assuming enzyme concentration stays the same Michaelis-Menten Plot 49  Limiting factor Limiting factor is enzyme is substrate Enzyme Kinetics 3 1 Vmax Low substrate concentration Steep increase in rate of reaction Rate of product formation is limited by concentration of available substrate Increased substrate concentration Rate of reaction levels off Enzymes totally saturated with substrate The rate of reaction reaches a maximum upper limit = maximum velocity (Vmax) What graph would you expect if reaction was not enzyme catalyzed? 3 enzymes active 1 enzyme active 0.5 sec to encounter substrate 5 enzymes active 1 sec to encounter substrate 1 sec to catalyze reaction 1 sec to catalyze reaction 1 sec to catalyze reaction = 1 product/1.5 sec = = 1 P/sec x 5 enzymes = 1 product/2 sec = 0.5 P/sec 0.67 P/sec x 3 enzymes = 2 P/sec = 5 P/sec 50 Vmax and Enzyme Concentration Rate of reactions increase for increased substrate and/or enzyme concentrations. But for any given enzyme concentration, there is a Vmax for that concentration 51  Measuring Vmax Michaelis-Menten Plot How quickly substrate consumed or product produced Keep everything COLD! Add all reagents/buffers and enzymes first Add substrate when you are ready to read – use plate reader and read all at once! 52 Dissociation Constant Kd The dissociation constant Kd measures the true affinity a ligand has for binding to an enzyme. A low Kd value = high affinity for binding to ligand 53 Michaelis Constant Km Km is the concentration of substrate which permits the enzyme to achieve half Vmax. This is a constant for the enzyme. Km is determined by the substrate’s dissociation constant (Kd) and how quickly the enzyme- substrate complex is turned over into product (kcat). Km 54 Why Are Km and Vmax Important? The lower the Km value for a given enzyme and substrate, the lower the [S] range in which the enzyme is effective A low value of Km is often interpreted as a high affinity of the enzyme for the substrate = reaction can reach ½ Vmax with a lower substrate concentration A large value for Km is often interpreted as a weak affinity of the enzyme for the substrate Vmax is important as a measure of the potential maximum rate of the reaction The rate at which a substrate will be converted to product once bound to the enzyme. By knowing Vmax, Km, and the in vivo substrate concentration, we can estimate the likely rate of the reaction under cellular conditions V = (Vmax [S])/(Km + [S]) 55 Km and Vmax Importance If two enzymes, in different pathways, compete for the same substrate, then knowing the values of Km and Vmax for both enzymes permits prediction of the metabolic fate of the substrate and the relative amount that will flow through each pathway under various conditions. Under what substrate concentrations would enzyme A be more active? Enzyme B? Enzyme A: Km = 0.01 mM/L; Vmax = 2 umol/min Enzyme B: Km = 0.1 mM/L; Vmax = 10 umol/min 56 Application to Km and Vmax Glucose enters cell and becomes phosphorylated to form glucose-6-phosphate. Phosphorylation has several functions Prevents glucose from leaving cell again Glucose has no charge, G-6-P is negatively charged Destabilizes glucose to facilitate further metabolism Glycolysis Two enzymes (isozymes) that can phosphorylate glucose Hexokinase and Glucokinase 57 Cellular Glucose Uptake 58 Application to Km and Vmax 2 Hexokinase Found in all tissues Insulin not required Inhibited by its own product G-6-P High affinity for glucose Low Km: 0.05 mM/L Operates efficiently at normal blood glucose levels (~4 mM) Adapted for utilizing glucose as an energy source Cellular respiration – ATP production FYI: Km for fructose = 6.7 mM/L 59 Application to Km and Vmax Glucokinase Found in liver, pancreas, (gut, brain) Activated by insulin and glucose Insulin increases transcription of GK gene in the liver Not inhibited by G-6-P Low affinity for glucose High Km: 5 – 6 mM/L Acts as a glucose sensor Regulates carbohydrate metabolism Response high when blood glucose levels high (after meal) Glucose stored as glycogen or fat High Vmax Liver effectively removes glucose from blood to prevent hyperglycemia 60 Question If blood glucose levels are very low (0.2 mM/L), which enzyme would phosphorylate more glucose? What if blood glucose levels were high (6 mM/L)? 61 Summary of Hexokinase vs Glucokinase 62 Turnover Number (kcat) kcat is the rate at which substrate molecules are converted to product by a single saturated enzyme at maximum velocity (moles of product/sec) The larger the kcat, the more product produced/sec Turnover numbers vary greatly among enzymes Low Km does not necessarily mean high kcat. Km High affinity could reduce rate of product release. 0.01 Catalysis might require other molecules that are present in limited amounts. 0.000005 Catalysis might require many steps. …. 63 Enzyme Efficiency Is a measure of how “efficiently” an enzyme converts substrates into products. Also, a measure of preference of the enzyme for the substrate The higher the number, the more the enzyme prefers this substrate Enzyme efficiency = kcat/Km Either a large value of kcat (rapid turnover) or a small value of Km (high affinity for substrate) makes kcat/Km large 64 Interpret the Km and kcat values Measured in molarity (CO2 to H2CO3) (Fumarate to Malate) 1. What does Km mean? 2. Which enzyme has the highest affinity for its substrate? The lowest? 3. What does kcat measure? 4. Which enzyme produces the most product/sec? Least? 5. Which enzyme is more efficient, carbonic anhydrase or fumerase? 65 Enzyme Efficiency 1 1. Look at carbonic anhydrase. Which substrate is the preferred substrate for this enzyme, CO2 or HCO3-? 2. Look at chymotrypsin. For which substrate does it have the highest affinity? Lowest preference? 66  ADH Gene Solve these problems 1. Which of the ADH isozymes have the highest affinity for ethanol? 2. Does it matter if you have ADH1B*1 or ADH1B*2? 3. Is ethanol broken down in the stomach? Would you say that the stomach is a major player in the catabolism of ethanol? 4. Where is most of the acetaldehyde metabolized, cytosol or mitochondria? ALDH Gene Km (mM) ALDH1 (cytosol) 0.03 5. How well would a person with ADH1B*2 and ALDH1 and ALDH2*2 ALDH2 (mitoch.)