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Lecture Material for Test 4.docx

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Department
Biology
Course
BIOLOGY 2F03
Professor
Lovaye Kajiura
Semester
Fall

Description
Lecture Material for Test 4 October 25, 2010 -memorize 20 aa -in orgo chem., more than 20 aa -only 20 in biological systems -some aa can be synthetically make in body, others must be obtained through food -intermediary metabolism: pathways to synthesize metabolic stuff… -hydrolysis cleaves peptide bonds in a polypeptide -alpha amino acid - have amino grp, carboxylic acid grip -R – 20 side chains -aa are chiral -amino grp/carboxylic group are ionizable at ph around 7.4 (carboxylate anion and ammonium cation) -zwitterion: overall neutral charge but has ions -charges changes properties Non polar -glycine: proton at side chain; not hydrophobic -alanine: methyl group; hydrophobic -valine: isopropyl group; sidechain has enough hydrophobicity that the aa is hydrophobic -usually in hydrophobic core, transmembrane domain Leucine: valine but has methlene group inserted Isoleucine: branch point at beta position Methionine: has sulphur (thioether bond) Proline: cyclic side chain Phenyalanine: flat aromatic molecular Tryptophan: largest Uncharged polar side chains Serine – alcohol can be ionized Threonine – variation of serine Asparagines – amide side chain Glutamine Tyrosine: phenol has resonization in benzene ring -tend to absorb electromagnetic energy (also tryptophan) Cysteine: like serine cept thio instead of alcohol` For Test Questions: Need to kno pka values in the table -only estimates compared to read world Amion acids with charged polar side chains Lysine – nitrogen -sometimes at enzymes, might have nH2 group with a lone pair (can fx as nucleophilic group) -usually with a nh4+ group Arginine – guanidinium ion -one of the last to have evolved in evolution Histidine: most versatile: immadazonium side chain -can fluctuate btw acid and base form -at the active site where proton transfers are occurring, this often positioned there Asparatic Acid -almost always in COOH- anionic form Glutamic Acid Peptidebond, condensation rxn -rxn involves loss of water molecule -carboxylic acid of one aa condensed with amino group of another aa to form amide bond -water produced in the process -amide btw two aa is called peptide -peptide bond is planar -terminus on right hand is C-terminus -N-terminus on the left -usually 100 aa per domain -usually in L formation -D aa present sometimes -synthesized as L then changed into D wwith enzyme -proteases are specific L amino acids -bacteria use D in the cell wall to escape proteases Isoleucine – has 2 chiral structures Threonine – R ? -oxidize 2 cysteines: disulfide bond -usually exposed to ECF to be in oxidized state -in the cytoplasm is usually reduced state Titration of Glycine Isoelectic point: pH at which the molecule is halfway being in negative form or positive form -take pK that flank the isoelectricpoint and average it -pI is where the net charge is 0 -pK depend on nearby groups -the only way to actually kno isoelectic point is to determine it experimentally since pK values depends on the microenvironment and fluctuates October 29, 2010 -all in R config except for cystein; biological isomer of cystein was S -all aa are in L config -threonine and isoleucine – chirality of sidechain of T is R and the chirality of the sidechain in I is S For L and R designation -groups in chiral centre projected towards us then assign chirality -quorum rule – take CO group go to R group, then the N group -If right, L amino -if left, R amino -enzymes are stereochemically specific -often get a mixture of 50 50 isomers when synthesizing drugs, but only need one of them (specifically) -ibuprofen: advil -thalidomide -women in early pregnancy given this to suppress nausea -biologically active isomer -other isomer caused birth defect Amino Acid Derivatives -actually more than 20 aa in nature -ribosome and translational machine only uses those 20 -enzymes modify aa residues after translation -these modifications determine where peptide/aa is designated (i.e surface) -centre carbon of aa is designated as alpha -bioillumination -caused my chemical rxn between aa in a protein -has conjugated pi system -post translational modification that occurs spontaneously -protein just folds into this conformation that allows chemical rxn to occur -protein becomes fluorescent after new conformation -absorbs radiation -can take this protein and fuse it to the gene of interest -then use this to follow gene in the subject through= fluorescence -important biological tool Biologically active aa -GABA – neurotransmitter -histamine – responsible for allergic reactions -dopamine – neurostransmitter (used for Parkinson’s) -thyroxine –produced by thyroid used for metabolism -oxidation of sulfhydral group for formation of sulfide bond only occurs of the aa on the way to ER since only then is oxygen available for the rxn -glutathione becomes oxidized after reduction of disulfide bonds that get formed spontaneously Chapter 5 Proteins: Primary Structure -only certain sequence selected by evol’n -structure drives selection of a particular sequence -primary: aa sequece plus any covalent bonds connected peptide together (i.e. disulfide bond) -intermolecular disulfide bonds between two chains of insulin -intramolecular disulfide bond – brings protein closer together -Test Q: if I treat insulin with reducing agent, what would happen to insulin after treatment with….. -secondary: how it folds -largest protein: titin -has 34000 aa, gigantic mRNA -cells that produce that involved in formation of muscle -two proteins that have same conformation with diff sequences will fold into distinct structures 2. Protein Purification and Analysis -assay protein: isolate protein -some proteins have diff isoelectric points and their solubility depends on isoelectric points so important to control pH -can use temperature if protein is temp-stable -proteolytic enzymes determine lifetime of protein -need to work fast, keep proteins on ice -can add inhibitors of proteolytic enzymes -adsorptive surfaces: some proteins become denatured when they get in contact with surfaces -surface tension -liquid nitrogen used to keep protein stable Assaying Proteins -coupled enzymatic activities: product of one rxn can be substrate of another -immunoassays -antibody to isolate protein -radioimmunoassay: when antibody is radioactive and can be visualized ELISA -nonspecific proteins -absorptive spectroscopy -can use specific dyes that particularly with proteins -coomassie blue: stain that reacts with protein and imparts blue colour to them -Bradfood assay: coomassie blue: binds to protein and shifts absorptive max November 1, 2010 Separation technique -at isoelectric point, protein least soluble -can add a salt to make it precipitate out -polarity – based on hydrophobicity -size – separates on basis on molecular weight -binding specificity – if has particular ligand, can sometimes link that ligand to resin -protein goes through column then can bind to ligand in resin and those that don’t bind travel through Salting out -using ammonium sulfate -a particular fraction of proteins precipitate -take that pellet, then centigfuge, then add more ammonium sulfate at higher concentration and repeat -way of purifying proteins -isoelectric point – point at which protein is least soluble -wide range -ion exchange chromatography -2 types of matrixes -diethyl aminoethyl cellulose – take negative proteins -resin packed into column -if it is positively charged, negatively charged proteins will bind, rest will wash out -vice versa for negative charged resin -proteins that have the same charge will elute first -can increase rate of elution by increasing salt gradient -salt neutralize chargers Gel filtration chromatography -protein placed in solvent on top of resin -smaller things are stuck -proteins that are largest are excluded from resin and elute first -largest proteins come out in void volume -small proteins that are able to interact with matrix -smallest interact the most and come out last -if protein has multiple subunit -MW can be determined by running through this column by a number of proteins of known MW Affinity chromatogprahy -need prior knowledge, background of protein -immuno affinity -sometimes antibody binds to much that it doesn’t let go -proteins that have 6 histidines have a natural affinity to nickel -can modify proteins to have this his-6-tag SDS-PAGE -separate protein without complex apparatus -detergent: SDS: can denature protein -anionic – interacts with protein and adds negative charge to protein -smallest proteins migrate the furthest -visulaize through dying -MW can be calibrated using a set of known weights -take protein and create gradient of pH in gel -add proteins to gel, turn on electric currnet -protein migrate to point where it coincides with its isoelectric point -then uses SDS in the other direction -shows realistically how many proteins are in a given cell -use this to show proteins that are expressed in a cell -can pick out spots on the gel and identify the protein Zonal ultracentrifugation -separate protein on buoyancy in sucrose gradient -was used to separate components of the ribosome -if not in the text.. then probably not on the test Protein Sequencing -sanger’s reagent: dinitrofluorobenzene -amino terminus gets labeled with yellow probe -hydrolyze protein by boiling it in 1m HCl -can break polypeptide chain into specific fragments -repeat process to get aa sequence by de
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