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Bio 2B03 Midterm 1 Studying Notes.docx

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Department
Biology
Course
BIOLOGY 2B03
Professor
Kim Dej
Semester
Fall

Description
Unit 1: Proteins Covers Slides from PPT 1 to 3-16. Sidechains differ in: size, shape charge, hydrophobicity and reactivity. They are classified by solubility in water or polarity of the side chain. Hydrophilic is charge polarized and capable of hydrogen bonding with water Hydrophobic are not polarized and unable to form hydrogen bonds with water, so water repels them in favor of bonding with itself. Proteins Made up of amino acids (20 kinds). By convention, the left end is N terminus, right end is C terminus. Proteins tend to have hydrophobic amino acids on the interior, to form a core. The protein conformation is the 3d arrangement of the polypeptide. Amino Acids A single amino acid consists of an alpha carbon attached to four substituents, H, carboxyl group, amino group and R group. They polymerize through peptide bonds which are reactions between the ionized states of amino acids undergoing dehydration synthesis. There are 4 special amino acids: Cysteine, Proline, Glycine and Histidine. They’re special properties are: ability to form disulphide bridges (covalent bonds), rigid structure forms kink, small size, shifts from positive/neutral on pH. Primary Structure The linear arrangement/sequence of amino acids, determined by the gene. A polypeptide of length n has 20 arrangements. Secondary Structure Conformation of a portion of the polypeptide resulting from folding of localized parts. Local interactions are non covalent bonds and include: Ionic bonds, hydrogen bonds, van der waal forces and hydrophobic effect. There are 3 major localized structures we observe: α-helix, β-sheet and turns/loops. The α-helix consists of carbonyl oxygens bonded to the amide nitrogen four residues towards the C-terminus, forming a rigid cylinder. In this arrangement the side chains are facing outwards and are responsible for hydrophobic/hydrophilic qualities. The β-sheet consists of laterally packed β strands (5-8 amino acids long) with H- bonds between carbonyl and amino groups of backbone in adjacent β strands. Forms independently of properties of side chains and has two arrangements; parallel and antiparallel. The R-chains determine the hydrophobic/hydrophilic quality of the surfaces of the sheets. β turns connect segments of β sheets and are 3-4 AA residues. Carbonyl of AA1 is H bonded to amino of AA4, proline at AA2 to introduce bend and glycine at AA3 to minimize steric hindrance. Motifs Motifs are particular combinations of secondary structures that recur in a variety of proteins and exhibit a particular 3d conformation associated with a particular function. Coiled-coil motif: 2 or more amphipathic α-helices wrapped around one another. Heptad (7) repeats with hydrophobic residues at position 1 and 4 of repeat. Found in DNA binding proteins. Zinc Finger motif: Consists of an α-helix and 2 β-strands held in position by the interaction of precisely positioned Cys or His residues with a zinc atom. Found in DNA binding proteins Helix-loop-helix motif: 2 α-helices joined by a loop region. Loop region can bind Ca 2+ or DNA via carboxyl side chains from Asp or Glu in the loop. Found in a vast number of Ca binding proteins. β barrel motif 4-10 antiparallel β-strands connected by hairpins each strand is successively added to the previous strand until the last strand is H bonded to the first strand to complete the barrel. Many proteins contain regions that lack a defined conformation; Intrinsically unstructured proteins which have a lack of tertiary structure as isolated subunits. Bioinformatic predict that a significant fraction of the genome codes for unstructured proteins, and that the fraction increases with the complexity of the organism. Unstructured proteins may acquire structure when associated in a complex. Or, unstructured proteins may assume specific folding patterns when associated with other molecules e.g. DNA associated with a Zinc-finger complex Tertiary Structure The overall conformation of a polypeptide; the fundamental unit of a tertiary structure is the domain. A domain is a substructure produced by any part a polypeptide chain that can fold independently into a compact stable structure. These are subdivided into 2 categories, structural and functional domains. Functional domains: regions of a protein that perform a certain activity Structural domains: regions of protein that form compact, largely independent globular domains Quaternary Structure Assembly of a multimeric protein; a functional protein composed of multiple polypeptides. Note prefixes such as homo/hetero/di/tri -mer. Post-translational Modifications Acetylation: Protects against intracellular protease degradation (80% of proteins) Methylation: Regulation of protein activity; methylation of histone tails (chromosome structure and gene regulation) Phosphorylation: covalent transfer of a phosphate group from ATP to the -OH group of serine, tyrosine, or threonine (kinases) to activate/deactivate proteins. Reversal of effect by removal of phosphate group (phosphatase) Hydroxylation: alter protein structure and function; animals - collagen - formation of triple-stranded helix; plants - cell wall proteins Glycosylation: Addition of carbohydrates/sugars.sugars added to -OH groups of serine and threonine. Many secreted and membrane proteins are glycosylated - this occurs in the Golgi apparatus. Allows for proper folding, protection from proteolysis, sensitization of responses of cell-surface proteins Carboxylation: allows binding of inorganic ions Lipidation: Addition of lipid molecule; anchors proteins to membranes. Prominent of cell signaling events. Protein Folding Polypeptides can begin to fold secondary and tertiary structures while being synthesized. Every polypeptide has a single lowest energy state, the native state. This is the most thermodynamically favorable state. Protein folding is spontaneous reversible and unique and all the information required for protein folding is contained in the primary sequence. In an experiment a 8m urea (breaks H & hydrophobic bonds) + mercaptoethanol (breaks disulphide bridges) was added to a folded protein. Dialysis removed the denaturants and the renaturation of the protein in vitro occurred spontaneously. Proof that information for protein folding lies in its sequence. Misfolded proteins Responsible for a variety of diseases, in 2 categories. Hereditary disorders and prion based diseases. If the protein is misfolded it may be in the wrong location to function, have no function or be detrimental to the cell. Prion: infectious agent that is protein based. A prion is thought to be a misfolded version of a functional protein where the misfolded protein binds to the regular protein and causes the regular protein to assume the abnormal conformation. Protein Folding Pathways Most proteins fold rapidly into their correct configuration. Incompletely folded proteins are helped to fold by chaperone proteins. Misfolded proteins are degraded. Chaperone Proteins There are two kinds: molecular chaperones and chaperonins. Molecular chaperonins selectively bind to hydrophobic amino acids that are exposed in the non-native conformation allowing it to fold correctly. HSP70 in cytosol and mitochondria, BiP in ER, DnaK in bacteria. HSP70 binds to hydrophobic elements to prevent aggregating and incorrect conformations from being formed, helping the polypeptide to get to the correct conformation. It only moves the protein into the right direction. HSP70 is produced in stressful environments, mostly heat. Functions by binding to protein, while hydrolysis of ATP to ADP to facilitate attaching. Protein folds a bit, then ATP rebinds to HSP70 causing it to release, protein finishes folding. Chaperonins form large cylindrical isolation chambers into which misfolded proteins are fed and folded within the chamber without interference from other macromolecules. TCiP cytosol, GroEL bacteria or chloroplast and Hsp60 in mitochondria. Assist in folding 15% of proteins (mammals). Misfolded protein is initially captured by hydrophobic interactions with rim of GroEL. ATP and cap (GroES) bind. Conformational change, space enlarges, releasing polypeptide into space and enclosing protein. ATP hydrolysis to ADP causes protein to eject. This ejection is a timed event, and the chamber does not interact with the protein. When ATP is bound, GroEL is in it’s relaxed conformation, however 1 is used per subunit, making it an energy intensive process. Protein Degradation Intracellular proteins are selected and marked for degradation by a protein degrading cellular machinery called the proteasome. Cells degrade: misfolded proteins, denatured proteins, too highly concentrated proteins, proteins taken up into the cell, and regulate levels of cyclic proteins. Tagging of the protein of ubiquitin molecules, a small protein with 76 residues. Degradation of the tagged protein into short peptides (7-8 residues) by the proteasome. Step 1: Ubiquitylation Addition of Ubiquitin to a protein targets that protein for degradation by proteasome. 3 Enzyme System E1 Ubiquitin activating enzyme E2 Ubiquitin conjugating enzyme E3 Ubiquitin Ligase: large family of protein each member recognizes a different signal Ubiquitin activated by linkage to E1, activated ubiquitin is transferred to cys on E2, E2 complexes with E3, E3 recognizes substrate and transfers ubiquitin to lysine side chain of target substrate. Protein is polyubiquitinated. Step 2 Proteasome Degradation. Proteasome = protein degradation machine. Once a polyubiquitin tag is attached, the protein is unfolded and then fed through one end of the proteasome cap, through the core, and it comes out the other. The core makes cuts at roughly at every 7-9 AA sequences,
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