Advanced Cell Bio Chapter 4
Advanced Cell Bio Chapter 4 BCMB 311
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This 7 page Class Notes was uploaded by Izabella Nill Gomez on Sunday February 7, 2016. The Class Notes belongs to BCMB 311 at University of Tennessee - Knoxville taught by Dr. Barry Bruce, Dr. J. Park in Spring 2016. Since its upload, it has received 13 views.
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Date Created: 02/07/16
Advanced Cell Bio Chapter 4 Notes! Proteins are the main building blocks form which cells are assembled and constitute most of a cell’s dry mass. Provide the cell with shape and structure; proteins execute nearly all its functions. Enzymes promote intracellular chemical reactions by providing intricate molecular sugars, contoured with bumps/crevices that cradle/exclude specific molecules. Proteins in the plasma form channels/pumps controlling nutrient passage; can also carry messages from one cell to another. Protein molecules are made from a long chain of amino acids, held together by covalent peptide bonds. Proteins are referred as polypeptides, and amino chains as polypeptide chains. In each type of protein, amino acids are present in a unique order (amino acid sequence)--same from one molecule of protein to the next. Each polypeptide has a backbone with a side chain--polypeptide backbone is formed form repeating a sequence of core atoms (-N-C-C-C-). The two ends of each amino acid are chemically different. The N-terminus carries an amino group (NH3+, NH2). C- terminus has carboxyl group (COO-, COOH). Projecting form the backbone are amino acid side chains--part of the amino acid that is not included in making peptide bonds. Give unique properties--can be nonpolar, hydrophobic, negatively/positively charged chemically reactive, etc. Long polypeptides are very flexible, as many peptide bonds that link C allow free rotation. The shape of folding is constrained by weak noncovalent bonds that form within proteins. Involve atoms in the backbone and side chains. Include H bonds, electrostatic attraction, Van der Waals. It takes many nthcovalent bonds to hold two regions of a polypeptide chain tightly together. The 4 weak intersection is hydrophobic, also has a central role in determining the shape a protein. In In an aqueous solution hydrophobes involving non-polar side chains tend to be formed together to minimize their disruptive effect on the H-bonded network of H2O. Important factor to folding of any protein is the distribution of polar/nonpolar amino acids. Nonpolar side chains (leucine, phenylalanine, valine, tryptophan) tend to fold to interior/polar side chains (arginine, glutamine, histone) interact to form H bonds. When polar amino acids inside protein, usually H bonded to other polar amino acids or the peptide chain backbone. Conformation: folded structure of polypeptide chain determined by what folding minimizes free energy (G)--releases heat and increases disorder (energetically favorable). Protein can be denatured by treatment with solvents that disrupt noncovalent interactions; converts protein into flexible polypeptide that’s lost its natural shape. Under conditions where the solvent is removed, folding occurs spontaneously (renaturation). Each protein normally folds into one stable conformation, but changes slightly with molecular interaction. Change of shape is crucial to function. When folded incorrectly, sometimes forms aggregates that damage cells (can cause neurodegenerative disorders--sheep (scapie)--(mad cow) cattle--(Jakob disease) human--caused by misfolded proteins--prions. Prions can convert good proteins to bad ones--considered infectious. Protein folding is usually assisted by chaperone proteins by shaping through energetically favorable pathways or through “isolation chambers” in which single polypeptides are few without the risk of forming aggregates in the crowded cytoplasm--chaperones make folding more efficient/reliable, but shape is specified by amino acid sequence. Proteins are the most structurally diverse in the cell-normally 50-2000 amino acids long. Hpr (bacterial transport cell) is 88 amino acids long, facilitates the transport of sugar into bacterial cells. Backbone model of protein illustrates the overall organization of protein; ribbon emphasizes folds, wire shows possible amino acids involved in activity; space filling reveals what amino acids are on the surface. Alpha-helix folding pattern was discovered in alpha-keratin (for skin, hair, etc.). Beta sheet is found in fibroin, a major component of silk. Both result from H bond from N- H and C-O groups in the backbone. Because the amino acid side chains are not involved, alpha-helices and beta sheets can be generated from different amino acid sequences. Causes repeating pattern to form. A helix is a regular structure resembling a spiral staircase. Generated by placing many similar subunits next to another in a strict repeating form. Depending on the twist, the helix can be right/left handed. The alpha helix is made when a single polypeptide chain turns around to form a cylinder. H bonds are made between every 4 amino acid, linking C-O of one bond to N-H of another. Gives rise to regular right-handed helix. Short regions of alpha helix are abundant in proteins embedded in cell membranes, such as transporters/receptors. Sometimes 2/3’s alpha helices wrap around one another to form a stable coiled-coil structure. Forms when alpha helices have most hydrophobic side chains on one side to twist around each other with side chains facing inward to minimize contact with the cytosol (ex: keratin, myosin). Beta sheet is made when H bonds form between segments of polypeptide chain that lie side by side. When neighboring segments run in the same orientation (like N to C terminus) structure is parallel beta sheet; if opposite, anti-parallel beta sheet. Both types produce rigid pleated structure and form one of many proteins. Beta sheets give silk its tensile strength; permit formation of amyloid fibers, an insoluble aggregate related also to Alzheimer’s. These structures are formed from abnormally folded proteins, stabilized by beta sheets that stack tightly with sides that chain like the teeth of a zipper. The primary structure of a protein is its amino acid sequence. The secondary includes alpha and beta sheets that form within the segments of the polypeptide chain. Tertiary is the 3D form (alphas, betas, random coils, any other loops between the N and C terminus). Quaternary involves a complex of more than 1 polypeptide. Protein domain is a level of organization; any segment of a polypeptide that can fold independently into a compact, stable structure (40-350 amino acids folded in alpha’s, beta’s, etc.)--molecular unit from which larger proteins are constructed. Different domains associate with different functions (ex: CAP< small domain to DNA, large to cyclic AMP). Larger proteins can have dozens of domains, connected by unstructured lengths of chain. These intrinsically disordered sequences are often found as short stretches of linking domains in otherwise ordered proteins--have lack of folded structure, do not form protein crystals. Unstructured sequences can wrap around one or more target proteins by binding with high specificity and low affinity--provide flexibility while increasing encounters between domains. Can help scaffold proteins bring together proteins in intracellular signal pathways. Also give proteins like elastin the ability to 20n form rubberlike fibers (recoiling after stretch). possible polypeptide chains possible from n amino acids. But a fraction are present due to stability and need for “behaved” ( no unwanted interactions=no aggregates) functional proteins. Protein families--one in which each family member has an amino acid sequence and 3D conformation that closely resemble the others. Ex: serine proteases (family of protein cleaving enzymes) include digestive enzymes, elastase, enzymes involved in blood clotting. Portions of their sequences are almost the same size--3D conformations are alike too. But enzymatic/cleaving activities affect different proteins. The same noncovalent bonds that allow specific protein also allow proteins to bind to each other to produce larger structures in the cell. A surface on a protein that interacts with another molecule through noncovalent bonds is a binding site. If the binding site recognizes the surface of a second protein, tight binding of 2 folded polypeptides will create a larger protein, one whose quaternary structure geometry is precise. Each polypeptide in this protein is a subunit that can have more than 1 domain. IN a simple case, 2 identical, folded chains from symmetrical copies of 2 protein subunits (dimer) are held by binding sites (ex: CAP protein). Other proteins do not have identical polypeptides (hemoglobin). Globular proteins are polypeptides that fold into a complex shape like a ball with irregular surface. Enzymes tend to be globular. Fibrous proteins generally have an elongated #D structure (when needed to span a large distance). One class ex: keratin filaments-- extremely stable. Alpha-keratin is a dimer with long alpha-helices forming coiled- coils capped at either end by globular domains with binding sites to assemble into ropelike intermediate filaments, Fibrous proteins are especially abundant outside the cell (extracellular matrix--binds cells to form tissue)--collagen is the most abundant--bound (overlapped together, they form collagen fibrils--extremely strong). Elastin is another fibrous protein, but loose and unstructured to form elastic fibers for lungs, skin, arteries to stretch and recoil. Many proteins are either attached to the membrane or secreted as a part of the extracellular matrix. To maintain structure, polypeptide chains are stabilized by covalent cross-linkages--can tie 2 amino acids in the same polypeptide or join many polypeptides in a large complex (ex: cell fibrils, elastic fibers). Most common covalent cross links are S-S bonds (disulfide bonds) formed before a protein is secreted by an enzyme in the ER that links 2 SH groups from cysteine side chains adjacent in folded proteins. They do not change conformation but act as an “atomic staple” to reinforce the most favored conformation. Ex: lysozyme retains antibacterial activity because it is stabilized by disulfide links. Generally do not form in cytosol--mild conditions. -Biological properties of a protein are determined by physical interaction with other molecules. All proteins stick/bind to other molecules in a specific manner. Sometimes tight/sometimes weak/short-lived. Binding has great specificity--any substance bound by protein is a ligand. The ability to bind selectively and with high affinity due to a set of weak, noncovalent interactions--requires many simultaneous bonds to hold--possible only if the surface contours of the ligand fit closely to the protein. When molecules match poorly, few noncovalent interactions occur, and the 2 molecules dissociate as rapidly as they come together. At the other extreme, noncovalent interactions occur and persist for a long time (ex: ribosome). Region of protein that associated with the ligand (binding site) belong to amino acids widely separated on linear polypeptide chain brought together when the protein folds. Other binding sites help regulate protein activity. Parts like alpha helices may be required to attract or attach a protein to a part of the cell. Antibodies are immunoglobin proteins produced by the immune system in response to foreign molecules, especially those on the surface of invading microorganisms. Each antibody binds to a particular target molecule extremely tightly, either inactivating it or marking it for destruction. Antibody recognizes the target--antigen with high specificity. Antibodies are Y-shaped molecules with 2 identical antigen binding sites, each complimentary to part of the antigen surface. Antigen binding sites are formed from several loops of the polypeptide chain that protrude from the ends of 2 closely juxtaposed protein domains. The amino acid sequence loops can vary greatly without altering the basic structure of the antibody. AN enormous diversity of antigen-binding sites can be generated by changing only the length of the amino acid sequence of loops (how a variety of antibodies are formed). Enzymes are responsible for nearly all of chemical transformations that occur in cells--proteins that need ligand binding as a first step for function. Enzymes bind to one or more ligands (substrates) and convert them into chemically modified products--enzymes speed up reactions without being changed (catalyst)--permit cells to make/break covalent bonds. Each enzyme is highly specific, catalyzing only a specific type of reaction. Ex: hexokinase adds a phosphate group to D-glucose but not to optical isomer L-glucose. Enzymes often work in tandem, with the product of one enzyme becoming a substrate for the next. Result is elaborate metabolic pathways. Lysozyme is an enzyme that acts as a natural antibiotic in egg white, saliva, tears, other secretions. Severs polysaccharide chains that form the cell walls of bacteria. Because of the bacterial cell being under osmotic pressure, a small number of polysaccharide chains that are cut ruptures the cell wall, bacteria bursts and lyses. The reaction catalyzed by lysozyme is hydrolysis--adds one molecule of H2O to a single bond between two sugar groups in a polysaccharide, causes the bond to break. The reaction is energetically favorable because free energy of the severed polysaccharide is lower than the free energy of the intact chain. There are energy barriers for catalyzed reactions, called activation energy. For colliding H2O molecules to break the bond linking the two sugars, the polysaccharide has to be distorted into a transition state in which atoms around the bond have altered geometrically and electron distribution is changed. Requires random molecular collisions for energy input. In aqueous solution at room temperature, the energy of collision almost never exceeds activation energy; therefore, hydrolysis occurs extremely slowly. This is where the enzyme comes in; binding site (active site) that cradles substrate. Because substrate is a polymer, lysozyme’s active site is a long groove that holds 6-linked sugars in the polysaccharide at the same time. As soon as the enzyme-substrate complex forms, the enzyme cuts the polysaccharide by catalyzing the addition of H2O to one sugar-sugar bond. Severed chain is released for further cleavage--active site of the enzyme contains precisely positioned chemical groups that speed up the reaction by altering the distribution of electrons in substrates. Binding to the enzyme also changes the shape of the substrate, bending bonds to drive to a certain transition state. Then, a brief covalent bond between substrate and amino acid side chain occurs in the active site. The final reaction leaves the enzyme unchanged. Many drugs used to prevent illness inhibit enzymes. Ex: cholesterol-lowering statins inhibit CoA reductase, involved in producing cholesterol. Some proteins need more than just amino acids to do its job; they employ non- protein molecules to aid. Ex: photoreceptor protein rhodopsin detects light by retinal (attached to the protein by covalent bond to the lysine side chain. Retinal changes shape to absorb a photon, leads to electrical signal by rhodopsin to the brain). Another ex: hemoglobin with 4 heme groups to pick up O2. Enzymes also use non- proteins (ex: biotin for enzymes--classified as vitamin, not found in body). Most proteins are regulated in coordination to maintain in optimal state--energy in cell not depleted. Regulation: 1. Amount of protein in a cell (regulates gene expression and degradation rate) 2. Control of enzymatic activities by confining enzymes in certain membranes 3. Protein level switches. Regulatory sites at enzymes alter the rate of the enzyme-substrate conversion. Feedback inhibition (negative regulation--prevents acting) has an enzyme acting early in the reaction inhibited by the late product in a pathway. In positive regulation, the enzyme is stimulated to produce. Active/regulatory site communicates to allow events at the active site to be influenced by binding at the regulatory site. Interaction between sites depends on the conformational changes in the protein. Many proteins are allosteric: can adopt 2 or more slightly different conformation and activity is regulated by the shift from one to another. Enzymes are regulated solely by the binding of small molecule. Another method of regulation is via phosphate groups, that causes major conformational change due to 2 negative charges by attracting positive side chains from somewhere in the same protein, affecting ligand binding. Reversible protein phosphorylation is used to control many proteins, can be caused by signaling intracellular/extracellularly. Involves the enzyme-catalyzed transfer of terminal phosphate group of ATP to hydroxyl group on a serine, theonin or tyrosine side chain of the protein; catalyzed by protein kinase. Reverse (dephosphorylation) catalyzed by protein phosphatase. Phosphorylation can either stimulate or inhibit protein activity. Phosphorylation can do more than control activity; can create a docking site where other proteins can bind. Promoting larger complexes. Phosphorylation is not the only form of covalent modification that can affect protein location/activity--can be affected by the addition of an acetyl group/lysine side chain. The addition of fatty palmitate to cysteine side chains drives protein to associate with the cell membrane. Ubiquitin can activate degradation. Eukaryotes have a second way of regulation for protein activity via P- addition/removal. P not enzymatically transferred, but part of guanine nucleotide-- GTP--bound tightly to types of GTP-binding proteins. These act as molecular switches--active when GTP bound, but can hydrolyze to GDP (removing a P) and inactivate. The process is reversible: conformational charges enable certain specific proteins to divide movements of cells and components=motor proteins generate the forces responsible for muscle contraction and most other movements. Also power intracellular movements of organelles/macromolecules (help move chromosomes and organelles). Many move via ATP hydrolysis to drive orderly conformational changes--can be rapid. From small, single-domain proteins to large proteins of many domains, functions that can be performed are elaborate. The most complex are carried out by assemblies formed from many molecules. DNA replication, gene transcription, protein synthesis, etc--catalyzed by highly coordinated linked set of many proteins---protein machines. Hydrolysis of ATP-GTP drives an ordered series of conformational changes in some of the individual protein subunits, enabling coordinated movement of proteins. In this way, enzymes can carry out successive reactions in order. Cell employ proteins machines to produce more efficient tasks products--better than doing it individually. To study, proteins are now isolated from lab-grown cells-“tricked” into making large quantities of protein using genetic engineering--the first step to the purification process is to break open the cells and release its contents--the result is cell homogenate/extract--a physical disruption followed by the initial fractionation procedure to separate the class of molecules of interest--ex: all soluble proteins. The job then is to isolate the desired protein. Involves purifying through chromatography steps that separate the mixture into fractions based on properties (sit, shape, electric charge). The most efficient is to separate the protein by the basis to bind to specific molecules--affinity chromosomes. Can also be separated by electrophoresis. Here, a mix of proteins is loaded onto a polymer gel and subjected to an electric field polymerase migrates on gel depending on size and net charge. If too big, can use 2D electrophoresis. To determine the amino acid sequence of a protein--can be done by directly analyzing amino acids ain the protein. First, protein is broken down due to a smaller process by using selective protease. Then countless amino acids determined chemically. First protein sequenced--insulin. Faster way is through mass spectrometry--determined the exact mass of every peptide in a purified protein, which allows the protein to be identified from a database that has a list of every protein though to be encoded by the genome of the organism in question. To perform mass spectrometry, peptides are derived from digestion with trypsin and are blasted with a lasers. Heats peptides, becoming electronically charged and ejected as a gas. Accelerated by the power of the electric field, peptide ions fly towards the detector, and the time is takes to arrive is related to the mass and charge. (larger-slower, les s mass, more charge). The masses of protein fragments are produced by trypsin cleavage serve as fingerprint to identify the protein and gene from databases. For more complex mistrusted, increased resolution can be found via tandem mass spectrometry--broken down by first mass spectrometer, then 2 nd3D conformation can be found via X-ray crystallography, nuclear magnetic resonance spectroscopy (NMR). With genetic engineering/biochemistry, mass production of proteins can be done and now proteins can be made to do unusual tasks, not as efficient as naturally occurring enzymes, but getting close.
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