Download Chapter 3 Notes Set 7

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SEQUENCING
The __________________ of a protein is defined as the sequence
(order) of amino acids in the protein chain from the N-terminal to
the C-terminal.
Sequence ultimately determines the function.
Sickle-cell anemia - caused by a single amino acid change from
that of normal hemoglobin.
_______ of proteins are _________________ - have
_______________________ in sequence within a population.
There are many normal human hemoglobins that vary in sequence
but the functioning of the protein is not affected.
Identifying the N-terminal
Can __________________________________ for detection
-
_________________________: highly fluorescent
_________________________: colored
_____________________________ (FDNB) (Sanger)
_____________________________ (PITC): used in
Edman degradation (sequencing)
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General scheme for sequencing of chains _____ amino acids long
1. _________________ the N-terminal amino acid ___________
2. _______ the derivatized N-terminal amino acid from the chain.
3. Physically separate the derivatized N-terminal amino acid from
the rest of the chain by _____________________.
4. Use a detection method to _______________________ of the
N-terminal amino acid.
_____________ is often used
5. _______________ with the rest of the chain from step 1 until
you reach the last amino acid.
See figure 3-25
Modifications for ______________________________
1. Break all __________________________.
2. Cut the chain into ____________ amino acids with an enzyme.
3. Separate the pieces and _____________ each piece separately.
4. ________steps 1,2 and 3 but using a ______________in step 2.
5. Compare results to determine how pieces fit together.
6. Use _______________to determine location of disulfide bonds
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________________________________
1. React with _______(phenylisothiocyantate) under mild alkaline
conditions
- form the PTC adduct (phenylthiocarbamyl derivative)
2. Add ___________ (lacking water)_______(trifluoroacetic acid)
- (analinothiazolinanone derivative)
- leaves the rest of the chain with 1 less amino acid
- _______________________________
- reaction does not degrade the rest of the chain
3. Selectively extract the anilinothiozolinone derivative into an
organic solvent
- physically separate the layers
4. Add ___________ to get the final PTH (phenylthiohydantoin)
derivative (more stable)
5. Identify the amino acid – PTH derivative by TLC, HPCL, GLC,
mass spectroscopy, etc.
6. Use the remaining chain and repeat from step 1 in cycles.
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For Chains Larger than 50 Amino Acids
1. Cleave the chain into pieces < 50 residues
________________– enzymes that ___________________
- usually a protein
- cleaves at the N or C terminal of specific residues
(don’t memorize, just be able to use this table)
Protease
Cleavage site
Trypsin
Lys, Arg ©
Submaxillarus protease
Arg (C)
Chymotrypsin
Phe, Trp, Tyr ©
Staphylococcus aureus
V8 protease
Asp-N-protease
Asp, Glu (C)
Pepsin
Phe, Trp, Tyr (N)
Cyanogen Bromide
(BrCN)
Met (C)
Asp, Gly (N)
 conditions need to be adjusted to maximize protease activity
 some are really more specific than others
 there are more besides the list above..............
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2. Break the disulfide bonds
Consider this oligopeptide that has a disulfide bond between
the cysteines:
Gly-Ala-Cys-Met-Val-Phe-Cys-Gly-Gly
S-S
What happens when you incubate with cyanogens bromide?
Gly-Ala-Cys-Met Val-Phe-Cys-Gly-Gly
S-S
But the two fragments are still joined by the -S-S- bond and
_______________________________________.
Disulfide bonds can be cleaved in several ways:
 _______________ by ____________________(HCOOOH)
Replaces S-S with 2 SO3-
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 Reduction with ____________ (____ or _______ reagent)
or with ________________________ (__-mercaptoethanol)
a. both cleave the -S-S- bond to give 2 -SH groups
DTT
2-mercaptoethanol
b. Disulfide bonds can spontaneously oxidize by _______.
Prevent by treating with ______________ (ICH3COO)
This________________________ the -SH groups.
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3. ___________________ by electrophoresis or chromatography.
4. _________________ each fragment by the Edman degradation.
5. ___________ at least one more time from the beginning with a
fresh sample of protein, but choose a ____________________.
Need this to put fragments in the proper order.
6. To finish the complete sequence, the location of S-S bonds
must be determined.
 use ______________________________________
- start with 2 fresh aliquots (portions) of protein
- treat one with a _________________
- treat other with the __________________ plus ______
- run samples side-by-side on a gel
- If no difference between samples  _________ present or
only –S-S- bonds between cysteines ___________________.
- If there is a difference –S-S- bonds are present.
Example:
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Importance of sequence determination
 ________________ to known sequences(databases) – is it new?
 Compare to ________________________________ (taxonomy)
 compare to proteins of ________________________
(__________________________ proteins)
- ____________ – homologous proteins in the ___________
-_____________ – homologous proteins from _____________
- find _________________ – for example the active site of an
enzyme – often highly similar from species to species
- 3 types of residues
1. _________________ – identical among homologs
2. _________________ – different among homologs
3. ________________ – some difference among homologs,
but substitutions are always with similar residues
(e.g. _____ <-> ______
or
_____ <-> ______ )
Example: cytochrome C (mitochondrial protein)
100 residues MW ~ 13,000
comparing 60 species  27 invariant
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Computer programs used to compare sequences and determine
relatedness and/or evolutionary history. Can weight importance of
substitutions with the ________ table (blocks substitution matrix).
Identify _________________________ – unique for the group
in which they are found (species, genus, etc.)
Synthesis of peptides:
Automated (so is sequencing)
Critical component is the _____________ at each reaction step.
Can synthesize a ______ amino long polypeptide in  few days.
A cell can do this in 5 sec!
1. Attach amino acid to a column resin.
______________ protects amino end from reaction
2. Remove Fmoc
3. Protect (Fmoc) and ______ (dicyclohexylcarbodiimide, DCC)
the next amino acid
4. Attach 2nd amino acid to one on the column
5. Repeat until peptide as long as desired
6. Use ______ to deprotect final amino acid and remove chain
from the column.
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