Ch 3 Text 3

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Author:
DesLee26
ID:
282308
Filename:
Ch 3 Text 3
Updated:
2014-09-07 09:17:05
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Sam
Folders:
Biochem
Description:
Test One
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  1. Advantages of knowing amino acid sequeces
    • The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist
    • Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathways
    • Amino acid sequences can be searched for the presence of internal repeats
    • Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing
    • Sequence data provide a basis for preparing antibodies specific for a protein of interest
    • Amino acid sequences are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins.

  2. o   The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist.

    CPUs do this in seconds. If the newly isolated protein is a member of an established class of protein, we can __
    begin to infer info about the protein's structure and function
  3. o   Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathways. Sequence differences give insight into __, especially if we assume the random mutation rate of proteins over time is __-->info about __
    • genealogical relationships
    • constant
    • when evolutionary lines diverged
  4. o   Amino acid sequences can be searched for the presence of internal repeats. These can reveal the __. Many proteins have arisen by __ of __ follwed by __.. 
    • history of an individual protein itself.
    • duplication of primordial genes followed by their diversification
  5. o   Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing. A protein destined for export from a cell or for location in a membrane contains a __, a stretch of about 10 hydrophobic residues near the amino terminus that directs the protein to the appropriate membrane. Another protein may contain a stretch of amino acids that functions as a __, directing the protien to __
    • signal sequence
    • nuclear localization signal, directing the protein to the nucleus
  6. o   Sequence data provide a basis for preparing antibodies specific for a protein of interest. One or more parts of the amino acid sequence of a protein will elicit an __when injected into a mouse or rabbit
    antibody
  7. o   Amino acid sequences are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins. Knowing primary structure enables __. DNA sequences can be determined from __ and can then be used as __to isolate the gene encoding the protein so that the __ can be determined. The gene then can provide valuable info about the __. __ is an integral part of molecular genetics, just as __ is central to the analysis of protein structure and function 
    • reverse genetics
    • amino acid sequences
    • probes 
    • entire sequence of the protein
    • physiological regulation of the protein
    • Protein sequencing
    • DNA cloning
  8. ·         Peptide sequences can be determined by __
    o   The first step: __

    • automated Edman degradation
    • determine the amino acid composition of the peptide, which is hydrolyzed into its constituent amino acids by heating it in 6 M HCl at 110oC for 24 hours.
  9. o   The amino acids can then be separated by __, where its identity is revealed by its __ (the volume of buffer used to remove the amino acid from the column) and its quantity is revealed by __
    • ion-exchange chromatography
    • elution volume
    • reaction with an indicator dye such as ninhydrin or fluroescamine
  10. o   After conjugation to the indicator, the amino acid exhibits a color with an intensity that is proportional to its __. A comparison of the chromatographic patterns of our sample __ with that of a standard mixture of amino acids would show that the amino acid composition of the peptide is (a particular set of amino acids)
    • concentration
    • hydrolysate
  11. o   The next step is to __
    §  Pehr Edman devised a method for __
    • identify the N-terminal amino acid
    • labeling the amino-terminal residue and cleaving it from the peptide without disrupting the peptide bonds between the other amino acid residues
  12. · The Edman degradation sequentially DOES WHAT?
    o   __ reacts with the __ of the peptide to form a __. Then, under mildly acidic conditions, a __ of the terminal amino acid is liberated, which leaves an __ shortened by __
    The cyclic compound is a __amino acid, which be identified by chromatographic methods
    • removes one residue at a time from the amino end of a peptide
    • Phenyl isothiocyanate
    • uncharged terminal amino group
    • phenylthiocaramoyl derivative
    • cyclic derivative
    • phenylthiohydantoin
  13. · The Edman procedure can then be repeated on the __, yielding another __, which can again be identified by chromatographic methods. Three more rounds of the Edman degradation will reveal the __
    o   By repeated Edman degradations, the amino acid sequence of some 50 residues in a protein can be determined
    • shortened peptide
    • PTH-amino acid
    • complete sequence of the original hexapeptide
  14. §  Gas-phase sequenators can analyze __quantities of peptides and proteins with the use of __ to identify each amino acid as it is released
    ·         This high sensitivity makes it feasible to do what?
    • picomole 
    • high-pressure liquid chromatography
    • analyze the sequence of a protein sample eluted from a single band of an SDS-polyacrylamide gel
  15. · Proteins can be specifically __ to facilitate analysis
    o   In practice, the peptides cannot be much longer than about __, because not all peptides in the reaction mixture do what at each step
    o   The peptides obtained by __ are separated by some type of __. The sequence of each purified peptide is then determined by the __. The order, however, is not yet known but obtained from overlap peptides
    §  A second enzyme is used to split the polypeptide chain at different linkages
    • cleaved into small peptides
    • 50 residues
    • release the amino acid derivative
    • specific chemical or enzymatic cleavage
    • chromatography
    • Edman method
  16. · Additional steps are necessary if the initial protein sample is actually __. __under reducing conditions should display the number of chains
    · Alternatively, the number of distinct __ could be determined. After a protein has been identified as being made up of two or more polypeptide chains, __ are used to dissociate chains held together by noncovalent bonds
    o   The dissociated chains must be separated from one another before sequence determination can begin
    • several polypeptide chains.
    • SDS-gel electrophoresis 
    • N-terminal amino acids
    • denaturing agents
  17. · Polypeptide chains linked by __ are separated by reduction with thiols such as __ or __. To prevent the cysteine residues from recombining, they are then __ with __ to form stable S-carboxymethyl derivatives. Then, sequencing can happen
    • disulfide bonds
    • beta-mercaptoethanol or dithiothreitol
    • alkylated with iodoacetate
  18. o   Genomic and Proteomic methods are complementary
    §  Thousands of proteins have been sequenced by the __ of peptides derived from specific cleavages. But, for sequencing proteins with more than 1000 residues, __—long stretches of DNA can be cloned and sequenced, and the nucleotide sequence can be translated to reveal the amino acid sequence of the protein encoded by the gene
    • Edman degradation
    • recombinant DNA technology is used
  19. §  Many proteins undergo __ after their syntheses; and, so DNA base sequences reveal the nascent protein before modifications
    · Some ends are __, others have __; __residues are oxidized to form __, side chains are altered, etc.
    • posttranslational modifications
    • trimmed
    • cleavage of a larger initial polypeptide chain
    • cysteine 
    • S-S links
  20. §  Amino acids have a lot of information, but don’t show modificationsà __

    · Thus, genomic and proteomic analyses are complementary approaches for __
    • chemical analyses
    • elucidating the structural basis of protein function

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