Amino Acids and Proteins: Nomenclature, Structure, and Separation, Quizzes of Biochemistry

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TERM 1 Building blocks of protein DEFINITION 1 Proteins are linear heteropolymers of a-amino acids and so are peptides that may or may not contain covalent modifcations such as lipids, carbohydrates etc. TERM 2 Amino acids properties DEFINITION 2 ___have properties that are well-suited to carry out a variety of biological functions Capacity to polymerize Useful acid- base properties Varied physical properties Varied chemical functionality TERM 3 What ways do proteins function as agents of biological function DEFINITION 3 Catalysis:Enolase (in the glycolytic pathway)DNA polymerase (in DNA replication)Transport of nutrients:Hemoglobin (transports O2 in the blood)Lactose permease (transports lactose across the cell membrane)Structure: Collagen (connective tissue) Keratin (hair, nails, feathers, horns)Motion at the cellular/intracellular level:Myosin (muscle tissue),Actin (muscle tissue, cell motility) TERM 4 Amino acids structure DEFINITION 4 Alpha carbon - want to identify firsthas amino group and carboxylic groupDiffer only at R substituent (side chain) TERM 5 Amino acid configuration L vs. D DEFINITION 5 If Amino acid is on LEFT --> L configurationIf amino acid is on RIGHT --> D configuration TERM 6 Amino acid properties similariteis and differences DEFINITION 6 Mosta-amino acidsare chrialalways has 4 subsittuents and is tetrahedralAll (EXCEPT PROLINE) have:An acidic carboxyl groupA basic primary amino groupAna-hydrogen connected to thea-carbon4th substiuent R is unique TERM 7 Glycine chirality DEFINITION 7 Achiral because it has two hydrgoens TERM 8 Amino acids: Atom naming Organic and Biochemical DEFINITION 8 Organic nomenclatrure: start from one endBiochemical designation-start froma-carbon and go down R group TERM 9 Nonpolar aliphatic R groups DEFINITION 9 Glycine-Gly- GAlanine-Ala -AProline-ProValine-ValLeucine- LeuIsoleucine-IleMethionineGAP-V-LIM TERM 10 Negatively charged R groups DEFINITION 10 Aspartate - Asp -DGlutamate -Glu- EDara Eats TERM 21 For amino acids w/out ionizable side chains, what is pI point DEFINITION 21 For amino acids w/out ionizable side chains, the isolectric point ispI = pKa1+pK2/ 2 TERM 22 When zwitterion, Amino acid is least soluble in water, why? DEFINITION 22 Because in the same molecule it has positive and negative charge that can interact w/ one another, do not need water TERM 23 When zwitterion, amino acid does not migrate in electric field, why? DEFINITION 23 Beause you need to split molecule apart in one direction or the other since it has both positive and negative charges TERM 24 How to calculate the pI when the side chain is ionizable DEFINITION 24 Identify species that carries a net zero chargeIdentify pKa value that defines the acid strength of zwitterion: pK2Identify pKa value that defines base strength of this zwitterion: pK1Take averag of two pKa valuesfor D & E, pI << 7, for K & R, pI >> 7 TERM 25 What are peptides, how do you form them, what happens after formed DEFINITION 25 Peptides are small condensation products of amino acidsTwo amino acids react, give up water and then you have an amide bond, no longer ionizable TERM 26 Peptides size vs proteins DEFINITION 26 are "small" compared to proteins (Mw<10kDa < 100 amino acids long) TERM 27 What is the only thing that matters in peptides and proteins? DEFINITION 27 Only the pKa's of the R groups matter TERM 28 Where does number and naming start and end for peptide DEFINITION 28 Numbering and naming starts from the amino terminus and ends at carboxyl terminal TERM 29 What will all peptides have no matter how long? DEFINITION 29 No matter how long peptide, will only hae one ionziable Nh3 group and the other end has carboxylate, everything else is an amide TERM 30 Functions of peptides and specific examples DEFINITION 30 Hormones and Pheromones- insulin (think sugar), oxytocin (think childbirth)Neuropeptides: substance P (pain mediator)Antibiotics: polymyxin B (Gram - bacteria), bacitracin (Gram + bacteriaProtection e.g. toxins: amanitin (mushrooms), chlorotoxin(scorpions) TERM 31 Proteins are DEFINITION 31 Polypeptides (covalently linked amino acids) + possibly: cofactor : functional amino acid component, metal ions or roganic molecules coenzymes: organic cofactors NAD+ in lactate deydrogenase prosthetic groups:covalently attached cofactors heme in myoglobin TERM 32 Polypetide size and number in proteins DEFINITION 32 Polypeptide size and number varies grealty in proteins TERM 33 Classes of conjugated proteins DEFINITION 33 lipoproteinsglycoproteinsphosphoproteinsflavoproteinsmetalloproteins TERM 34 Protein Biochemistry DEFINITION 34 Every protein in cell has a particular functionstructure of a protein determines its functionamino acid sequence of protein determines it structure TERM 35 How do you determine sequence or structure or activity of protein DEFINITION 35 in order to determine sequence or structure or activity of protein, you need purified proteinw. purified protein, you can determin its amino acid sequence, its structure, and characterize is activity TERM 46 What will be the charge of basic proteins at very high pH (>12)? At very low pH ( DEFINITION 46 Take proteins to extreme pHs, denature it,highly dependent on pH TERM 47 What does the charge of the protein depend on? DEFINITION 47 The ____ not just depends on the types and # of acidic/basic side chains but also on the pH TERM 48 Separation by charge (contains, mechanism, naming) DEFINITION 48 column resin is oppositely charged species of wanted protein, Separation is based on differences in sign and magnitude of charge on a proteinBinds charged proteinsMore the net +ve or =ve charge on protein --> binds more tightly to columnIf protein is negatively charged = anion exchange, of protein is positively charged = cation exchange TERM 49 separation by charge elution DEFINITION 49 1.Compete it off with another cation i.e. Sodium Chloride high concentration, NaCL going to compete with protein and bind to resin, displace protein, preferred method 2. Change the charge of the protein itself, change the pH, INCREASE the pH, hyodrxyl charge negatively charged, stability of fold of amnio acid will decrease if pH taken beyond 10 TERM 50 Separation by size (called what,contains, mechanism) DEFINITION 50 called size - exclusion chromatography, also called gel filtrationcolumn contains resin that has multiple pores/cavities distributed all overMixture of different size proteins added to top of column and buffer is run through columnBigger proteins too big to enter cavities --> come out firstSmaller proteins enter the cavities, take more convoluted path --> elute later TERM 51 Separation by size (detection, collection, Ve and V0) DEFINITION 51 purified samples detected using UV detector and collected in fractionsVe = specific elution volume of proteinV0 = void volume (exclusion volume) of column (constant for given column)Relative elution volume Ve,/V0 depends on molecular mass)Larger the molar mass, the lower the VeRelative elution volume Ve/V0 depends on molecular mass TERM 52 Separation by Affinity (contains, mechanism, elution) DEFINITION 52 separation is based on bindingchromatography column has resin that is covalently attached to a chemical molecule called a ligandligand is selected such that it has high binding affinity to a specific protein of interestprotein of interest binds to ligand in column while resto f portines move freely.protein eluted from column using special buffer that disrupts ligand-protein interaction, sometimes by means of free ligand which protein binds more to TERM 53 Three common types of Affinity chromatography (what is needed) DEFINITION 53 All three types require covalent tags attached to protein of interestNi+2 column binds polyhistidine tagGlutatione column binds glutathione S-transferase (GST) tagAmylose column binds maltose bidning protein (MBP) ta TERM 54 What are the ways to analyze proteins? DEFINITION 54 SDS PageIsoelectric focusing2D-PAGEActivity experimentsElectrophoreis TERM 55 Electrophoresis DEFINITION 55 Electric field pulls proteins accoding to CHARGEgel matrix hindres mobility of proteins according to their size and shapeBuffer on top, buffer on bottom in additional to meshAnything thats negatively charged will move down, positive charge at bottom TERM 56 How can we make sure mobility of proteins in electrophoresis depend just on size? DEFINITION 56 SDS Page TERM 57 What is SDS, what does it do? DEFINITION 57 SDS = sodium dodecyl sulfate - ionic detergentSDS micelles bind to and denature/unfold all proteins-SDS gives all prteoins uniformely negative chageAll proteins denatured - lose their native shape Surround them completely with SDS that makes the SDS charge (negatively charged)The SDS binds uniformly per unit length of protein and therefore the force on the molecules from the field will be a uniform amount per unit length, and the only affect on the speed of travel will be the retarding force due to their size. TERM 58 How do we visualize the proteins on the gel DEFINITION 58 Coomassie blue staining - used to visualize all proteins in a mixture (because it binds to arginine, histidine and aromatic amino acids)Western blot (or immunoblot) - to visualize only a specific protein TERM 59 SDS-Page and molecular weight DEFINITION 59 SDS page can be used to calculate molecular weight of a proteinalways have to have molecular weight markerscan compare to standard proteins of known molecular weight TERM 60 Western Blot disadvantages DEFINITION 60 1. Proteins separated by SDS-PAGE (DON"T do 2. transferred onto nitrocellulose membrane3. Antigen with antibody bind to protein4, Secondary antibody reacts with first antibody5. Secondary carries label that can be detected (flourescent)Need to know a lot about protein in order to make antibody