Biochem Protein Lecture 5

Question 1

The first amino acid in the sequence is called the

The last aa in the sequence is called the

Answer 1

Amino (N-terminal)

Carboxyl (C-terminal)

Question 2

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

Answer 2

Insulin

primary structure

Disulfide

Question 3

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

Answer 3

!

Question 4

Disulfide bonds can be both inter and intrachains.

Answer 4

!

Question 5

nsulin has two different chains. Why?

Answer 5

–N-terminal analysis of insulin indicates the presence of both gly and phe.

–C-terminal analysis indicates the presence of both asn and ala

Question 6

The folowing reagents react with the N-terminal amino group of the polypeptide chain

Answer 6

Sanger’s Reagent
Dansyl Chloride
Dabsyl Chloride
Edman’s Reagent

Question 7

When Sanger’s reagent (DNF) is reacted with the N

-terminal amino group of a polypeptide in alkaline solution, a ______ is formed

Answer 7

DNP derivative

Question 8

In the Sanger method aqueous acid hydrolysis of the ____ bonds destroys the polpeptide

Answer 8

Peptide bonds

Question 9

Be able to recognize dansyl and dabsyl, know structure of Sanger’s reagent

Answer 9

Womp.

Question 10

Can the Sanger method be used to sequence a polypeptide beyond its N-terminal residue?

Answer 10

No

Question 11

Can the Sanger method be used to determine the number of polypeptides in a protein?

Answer 11

Yes, as long as each chain has a DIFFERENT N-terminal residue

Question 12

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

Answer 12

Edman Degradation

Anhydrous trifluoroacetic acid

Question 13

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

Answer 13

Acid

Chromatographic

Question 14

Unlike the Sanger Method, what does Edman’s Degradation do?

Answer 14

Leaves the rest of the polypeptide chain intact.

Question 15

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?

Answer 15

Yes

Yes, for shorter peptides

Question 16

The C-terminal amino acid can be determined by enzymatic hydrolysis with a

Answer 16

carboxypeptidase

Question 17

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

Answer 17

50aa

Question 18

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

Answer 18

Specificities

arg, lys, or pro.

pro

Question 19

carboxypeptidase are _______; they cleave the C-terminal peptide bond

Answer 19

Exopeptidases

Question 20

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

Answer 20

Hydrolyzed

at which the aa is released from the chain

Question 21

Rn-1 means the AA to the ____ of the C-terminus

Answer 21

Left

Question 22

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 _______

Answer 22

Hydrazinolysis

C-terminal aa

Sequencing

Question 23

Slide 11

Answer 23

!

Question 24

What are the two ways disulfide bonds would be broken in the insulin example?

Answer 24

Adding an oxidizing agent

Adding a reducing agent

Question 25

The mercapto group of cysteine must be derivatized to prevent reoxidation when reducing disulfide bonds to form cys residues, what is frequently used?

Answer 25

Iodoacetate

Question 26

This method of breaking disulfide bonds produces two cysteic acid resiudes

This method forms cys residues

Answer 26

Oxidizing Agent

Reducing Agent

Question 27

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

Answer 27

Electrophoresis

Ion Exchange Chromatography

Question 28

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

Answer 28

Proteases

Question 29

Enzymes that catalyze the hydrolytic cleavage of peptide bonds

Answer 29

Proteases

Question 30

What does trypsin cleave?

Chymotrypsin?

Cyanogen bromide?

Answer 30

Lys, Arg (C)

Phe, Trp, Tyr (C)

Met (C)

Question 31

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

Answer 31

!

Question 32

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

Answer 32

3. Huh?

Chromatography or electrophoretic methods

Edman procedure

Question 33

If arginine is the end of a fragment, and trypsin is used for cleaving, you would have one less fragment? Ask about this

Answer 33

!

Question 34

Cleaving proteins bonds with ______ check for disulfide bonds in the example.

How?

Answer 34

trypsin

It would cleave RR groups but there would be no fragment due to disulfide bonds keeping them together

Question 35

What molecule can be used to check for free sulfide bonds (unbonded disulfide bonds)

Answer 35

iodoacetate

ICH2COO(-)

Question 36

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

Answer 36

CNBr or an endopeptidase

Question 37

What are the other methods researchers use for determining aa sequences

Answer 37

Mass spectrometry (20-30aa resiudes)

Or they can determine the sequence of nucleotides in the gene that codes for it