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