Biochem Protein Lecture 5 Flashcards
The first amino acid in the sequence is called the
The last aa in the sequence is called the
Amino (N-terminal)
Carboxyl (C-terminal)
The primary structure of ____ is an example frequencly used for determining protein ___ structure
Many proteins, like insulin, contain more than one chain. Also, in some proteins, polypeptide chains are covalently linked by ___ bonds between cys residues
Insulin
primary structure
Disulfide
In the first step of primary structure determination, the number of different polypeptide chains in the protein is determined. Identifying the N-and C- terminal amino acids determines the number of different chains
!
Disulfide bonds can be both inter and intrachains.
!
nsulin has two different chains. Why?
–N-terminal analysis of insulin indicates the presence of both gly and phe.
–C-terminal analysis indicates the presence of both asn and ala
The folowing reagents react with the N-terminal amino group of the polypeptide chain
Sanger’s Reagent
Dansyl Chloride
Dabsyl Chloride
Edman’s Reagent
When Sanger’s reagent (DNF) is reacted with the N
-terminal amino group of a polypeptide in alkaline solution, a ______ is formed
DNP derivative
In the Sanger method aqueous acid hydrolysis of the ____ bonds destroys the polpeptide
Peptide bonds
Be able to recognize dansyl and dabsyl, know structure of Sanger’s reagent
Womp.
Can the Sanger method be used to sequence a polypeptide beyond its N-terminal residue?
No
Can the Sanger method be used to determine the number of polypeptides in a protein?
Yes, as long as each chain has a DIFFERENT N-terminal residue
In this procedure,the amino -terminal residue is labeled with phenylisothiocyanate at pH 9 to yield a phenylthiocarbamoyl (PTC) -peptide.
This derivative is treated with _______ to remove the amino terminal labeled residue from the rest of the polypeptide as an anilinothiazolinone derivative
Edman Degradation
Anhydrous trifluoroacetic acid
The anilinothiazolinone deriviative after the second step of Edman’s degradation is treated with aqueous ________ to convert it to a more stable adduct called a phenylthiohydantoin derivative
The PTA-aa is identified by comparing its ______ properties to known PTH-aa derivatives
Acid
Chromatographic
Unlike the Sanger Method, what does Edman’s Degradation do?
Leaves the rest of the polypeptide chain intact.
In Edman’s, after treating the first AA residue, the rest of the polypetide chain can be treated with Edman’s reagent to yield the second AA in the sequence.
Can it be used to sequence an entire polypeptide?
What about an entire sequence?
Yes
Yes, for shorter peptides
The C-terminal amino acid can be determined by enzymatic hydrolysis with a
carboxypeptidase
Edman’s Degradation has been automated using a machine called a sequenator; the sequence can be determined up to ___ aa away from the N-terminal end
50aa
Different carboxypeptidases have different _____
For example, carbozypeptidase A will not cleave the C- terminal residue if it is
Also, carboxypeptidase A won’t cleave any aa if the next to last aa,Rn-1, is
Specificities
arg, lys, or pro.
pro
carboxypeptidase are _______; they cleave the C-terminal peptide bond
Exopeptidases
The cleavage of the C-terminal aa results in a
shorter polypeptide chain with a new C- terminal residue; it may be subsequently
The original C-terminal aa must be determined by following the rate at which the _______________. Aliquots from the solution are removed at various times and analyzed for the amino acid
Hydrolyzed
at which the aa is released from the chain
Rn-1 means the AA to the ____ of the C-terminus
Left
The original C-terminal can also be determined by this method, which is a reaction of a polypeptide with hydrazine leading to aa hydrazides of all the aa except the _________
After determining the number of polypeptide chains in the protein via N and Cterminal analysis, different chains are separated from each other for _______
Hydrazinolysis
C-terminal aa
Sequencing
Slide 11
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What are the two ways disulfide bonds would be broken in the insulin example?
Adding an oxidizing agent
Adding a reducing agent
The mercapto group of cysteine must be derivatized to prevent reoxidation when reducing disulfide bonds to form cys residues, what is frequently used?
Iodoacetate
This method of breaking disulfide bonds produces two cysteic acid resiudes
This method forms cys residues
Oxidizing Agent
Reducing Agent
After breaking disulfide bonds, the mixture of chains could be separated by ___ or _____; differences in pIs of the different chains would permit this kind of analysis
Electrophoresis
Ion Exchange Chromatography
To determine the sequence of a polypeptide chain after breaking disulfide bonds and separating them, samples of the polypeptide chain are separately cleaved into shorter peptide fragments using different chemical reagents or
Proteases
Enzymes that catalyze the hydrolytic cleavage of peptide bonds
Proteases
What does trypsin cleave?
Chymotrypsin?
Cyanogen bromide?
Lys, Arg (C)
Phe, Trp, Tyr (C)
Met (C)
Note that proteases that cleave at the C terminus end it ends on that aa,
if it cleaves at the N terminus end of an AA, the aa before that is the last one
!
consider the enzyme trypsin. It catalyzes the hydrolysis of peptide bonds in which the carbonyl group is either an arg or lys residue.
Therefore, a polypeptide with 3 arg and/or lys residues usually results in four smaller peptides on cleavage with trypsin. All residues, except one, will
have a carboxy-terminal lys or arg
If the same polypeptide were separately hydrolyzed, the total number of lys and arg residues in the original polypeptide would be ___
The fragments produced by trypsin would be separated by _______ or ______ methods and sequenced by
- Huh?
Chromatography or electrophoretic methods
Edman procedure
If arginine is the end of a fragment, and trypsin is used for cleaving, you would have one less fragment? Ask about this
!
Cleaving proteins bonds with ______ check for disulfide bonds in the example.
How?
trypsin
It would cleave RR groups but there would be no fragment due to disulfide bonds keeping them together
What molecule can be used to check for free sulfide bonds (unbonded disulfide bonds)
iodoacetate
ICH2COO(-)
To determine which disulfide bond arrangement is present, a sample of protein must be treated with: _______ or an _____ to obtain fragments with the disulfide bonds intact
If the two chains are cleaved between the
cys residues, one can determine which cys residues form the disulfide bonds
CNBr or an endopeptidase
What are the other methods researchers use for determining aa sequences
Mass spectrometry (20-30aa resiudes)
Or they can determine the sequence of nucleotides in the gene that codes for it
