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Biochemistry
test 2 Proteins,
| Question | Answer |
|---|---|
| What are the roles of proteins? | 1. regulatory, 2. monitor extra/intra cellular conditions, 3. relay info to other cellular components |
| Proteins are essential structural components of cells? | TRUE |
| protiens can be classified as? | primary, secondary, tertiary, and quaternary structures |
| what makes up the primary structure of a protein? | amino acid sequence of the polypeptide chain/chains |
| 2ndary, 3iary, 4ternary refer to ? | 3D shape of folded polypeptide chains |
| proteins are synthesized in vivo? in vitro? | IN VIVO |
| how are proteins synthesized in vivo? | stepwise polymerization of AA in correct order |
| for a protein of n residues ho wmany possible sequences are there? | 20^n |
| polypeptides smaller than 40 residues are called ____ | peptides |
| what is the largest known polypeptide chain? | titin. 26,926 residues |
| factors considered when purifying proteins | pH, Temp, presence of degradative enzymes, adsorption to surfaces, Long-term storage |
| what is normal Temp for purifying protein? | 0 degrees C |
| 2 degradative enzyme types | proteases, nucleases |
| how can degradative enzymes be inhibited? | adjusting pH/Temp, or by adding compounds that block their action |
| when dealing with long-term storage what are 2 concerns that must be prevented? | slow oxidation/microbial contamination (nitrogen/argon gas can help prevent this) |
| the rate of product formation is proportional to the amount of enzyme present? | TRUE |
| coupled enzymatic reaction is? | page 99 |
| what are immunoassays? | antibodies, proteins produced by an animal's immune system in response to introduction of a foreign substance |
| 2 techniques for detecting proteins | 1. RIA radioimmunoassay, 2 ELISA enzyme-linked immunosorbent assay |
| how can the [] of a substance in a solution be measured? | absorbance sperctroscopy |
| whaqt is the beer-lambert law | A= log( I0/I)= ecl, A=absorbance, I0= intensity of incident light at given lambda |
| if a protein has a chromophore that absorbs in the visible region of the spectrum then the absorbance can be used for what? | to assay for presence of this protein in am ixture of other proteins |
| solubility of a protein depends on [] of what? | dissolved salts, polarity of solvent, pH, and Temp |
| what is salting in? | phenomenon where solubility of a protein at low ion [] increases as salt is added |
| what is salting out? | result of competition betw added salt ions and the other dissolved solutes for molecules of solvent |
| what does HPLC (high performance liquid chromatography) | it employs automated systems with precisely applied samples, controlled flow rates at high pressures, a chromatographic matrix of specially made 3-300 microdiameter glass or beads coated with a material, and online sample detection |
| what happens in ion exchange chromatography? | charged molecules bind to oppositely charged groups that have been immobilized on the matrix |
| andions bind to cationic groups on ___? | anion exchangers |
| the most used anion exchanger is a matrix attached with ___? | DEAE deithylaminoehtyl |
| most used cation exchanger has ___? | CM carboxymethyl groups |
| what are polyelectrolytes? | polyionic polymers |
| the binding affinity of a particular protein depends on? | the presence of other ions that compete with the protein for binding to the ion exchanger and on the pH of the solution, which influences the net charge of the protein |
| look at page 102 for steps in | yup |
| proteins that can bind tightly to a ion exchanger can be..... | eluted |
| what is eluted? | washed through the column |
| what is an eluant? | a buffer that has a higher salt [] of a pH that reduces the affinity with which the matrix binds the protein |
| in you substitute the matrix material with octyl or phenyl groups then youa re using what kind of chromatography>? | hydrophobic interaction chromatography |
| what is used in metal chelate affinity chromatography? | a divalent metal ion like Zn2+ or Ni2+ is attached to the chromatographic matrix so that proteins that have metal-chelating groups can be retained. |
| His-Tag | His residues at a N or C terminus of a polypeptide |
| what is Electrophoresis? | migration of ions in an electric field |
| what is the differecne in electrophoresis from gel filtration? | electrophoretic mobility of smaller molecules is greater than the mobility of larger molecules with same charge density |
| what is immunoblotting or western blotting? | process used if an antibody to a protein i savailable and can be used to specifically detect this protein on a gel in presence of other proteins |
| what is the purpose of 2-mercaptoethanol? | reducing agent that breaks disulfide bonds |
| what does Capillary electrophoresis (CE) do? | uses thin capillary tubes that rapidly dissipate heat. used to separate only small amounts of material. |
| IEF isoelectric focusing` | deals with pI and each species of protein is focused into a band about its pI |
| 2D gel electrophoresis | IEF combined with SDS-PAGE |
| up to 5000 proteins have been observed on a single 2D electrophoretogram | TRUE |
| what is a valuable tool for PROTEOMICS? | 2D gel electrophoresis |
| what is proteomics? | involves cataloguing all of a cell's expressed proteins with emphasis on their quantitation, localization, modifications, interactions, and activities |
| what is the analytical ultracentrifuge used for today? | characterizing systems of noncovalently associating molecules |
| what happens in preparative ultracentrifugation? | sedimentation is calrried out in solution of inert subst. in which [] and the density increase from top to bottom of the centrifuge tube |
| what happens in Zonal ultracentrifugation? | MACROmolecular solution is layered on top of preformed density gradient (usually SUCROSE) |
| equilibrium density gradient centrifugation | look at page 108 |
| what was the 1st protein sequence of? | bovine insulin |
| who reported the bovine insulin? | Frederick Sanger |
| what yields with primary amines to yield dansylated polypeptides? | dansyl chloride |
| what temp is needed for dansyl chloride treatment? | high temperatures |
| what is mercaptan? | what is used to cleave disulfide bonds between Cys residues |
| free sulfhydryl groups are treated with ___ to prevent re-formation of disulfide bonds through oxidation? | iodoacetate |
| how are disulfide bonds created? | oxidation |
| what is amino acid composition? | number of each type of amino acid residue present |
| how is the amino acid composition of a polypeptide determined | complete hydrolysis |
| acdi hydrolysis degrades what? | Ser, Thr, tyr, trp |
| base hydrolysis destroys what? | Cys, Ser, Thr, and Arg |
| what does a protease do? | cleave peptide bonds |
| trypsin cleaves | carboxyl side of Arg and Lys |
| edmans reagent reacts with an N amino group to make | PTC phenylthiocarbamyl |
| what is mass spectrometry | measures mass to charge ratio for ions in gas phase |
| ESI | electrospray ionization |
| short polypeptides <25 residues can be directly sequenced through use of | tandem mass spectrometer |
| Darwinian evolution | a mutation that arises and improves fitness of host |
| what protein is found in nearly all eukaryotes? | cytochrome c |
| homologous proteins? | evolutionarily related proteins |
| invariant residue | a residue essential to that proteins function |
| hypervariable | position of a residue that is not function specific |
| neutral drift? | process by which random nature of mutational processes will in time change a protein in ways that will not significantly affect its function |
| hypervariable residues are particularly subject to neutral shift | TRUE |
| distances between branch points are expressed as? | # of amino acid differences per 100 residues of the protein |
| plot for protein is linear indicating.... | mutations accumulate at constant rate over time |
| what is one of the most highly conserved proteins? | Histone H4 |
| why is gene duplication efficient? | bc one copy of the gene evolves a new function through natural selection |
| 2 independently evolving genes that are derived from a duplication event are? | paralogous |
| what is faster? generating new genes by shuffling modules? or duplicating an entire gene and allowing it to mutate over time? | generating new genes by shuffling modules |
Created by:
jlvon425
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