Biochemistry 153A Protein Structure Study Questions Page 1

Biochemistry 153A Protein Structure Study Questions

Introduction to Protein Structure

1. Provide the appropriate term for each of the following: (a) Protein with nucleic acid prosthetic group (b) Protein with metal ion prosthetic group (c) Protein with more than one polypeptide chain (d) One polypeptide of a protein having more than one polypeptide chain (e) Amino acid sequence of a protein (f) Three dimensional folding of a polypeptide chain (g) Destruction of the three-dimensional structure of a protein 2. Briefly indicate how increasing the concentration of ammonium sulfate [(NH4)2SO4] leads

to the aggregation and precipitation of a subset of a mixture of proteins. 3. Briefly describe the principle(s) by which isoelectric focusing can be used to separate

proteins. Provide an example of a specific application of this method described in class.

Primary Structure of Proteins

1. Diagram the mechanism of acid hydrolysis of a peptide bond and indicate the problems associated with using acid hydrolysis to determine the amino acid composition of a peptide.

2. Identify the following molecule and explain its use in determining the primary structure of proteins.

O2N F

NO2 3. Briefly describe the general principles of the Edman Degradation method of amino acid

sequencing. Do not attempt to describe the individual reaction steps, but do identify the Edman reagent and any products analyzed in the method.

4. Indicate the reaction conditions and the final products involved in the Edman degradation.

N C S H2N C C

R1

H

NH

O

+

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5. Draw the products of cleaving the following peptide with (a) trypsin or (b) cyanogen bromide.

+H3N CH C

CH3 O

NH CH C NH

OCH2

CH2

CH2

CH2

CH C

CH2

CH2

S

CH3

NH CH COO–

O CH2

NH3+

(c) Name the tetrapeptide (3-letter code for amino acids). (d) How many different structures are possible having this composition? 6. What are the product(s) of the oxidation of disulfide bonds with performic acid? Identify

the purpose of this reaction. 7. A peptide containing 10 amino acids was analyzed and found to contain the following

amino acids after complete hydrolysis (listed alphabetically): Ala Arg Gly Ile Leu Lys Met Phe Ser Val Treatment of the intact peptide with 2,4-dinitrofluorobenzene, followed by acid

hydrolysis, yielded 9 amino acids and the following compound: NO2

O2N NH C

CH2CH(CH3)2

H

COOH

Treatment with the indicated reagents yielded peptides with the indicated

composition: Cyanogen bromide: CB-1: Arg, Ile, Leu, Met CB-2: Ala, Gly, Lys, Phe, Ser, Val Chymotrypsin: CT-1: Gly, Ser CT-2: Ala, Arg, Ile, Leu, Lys, Met, Phe, Val Trypsin: T-1: Arg, Ile, Leu T-2: Gly, Ser, Phe T-3: Ala, Lys, Met, Val

Carboxypeptidase treatment of the intact peptide liberated a nine-amino acid peptide and serine.

Using these experimental observations, indicate the two possible sequences of this peptide. How might you distinguish between these alternatives?

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8. A peptide containing 10 amino acids was analyzed and found to contain the following amino acids after complete hydrolysis:

Ala Arg Gly Ile Leu Lys Met Phe Ser Val Treatment of the intact peptide with 2,4-dinitrofluorobenzene, followed by acid hydrolysis, yielded 9 amino acids and the following compound:

NO2

O2N NH C

CH2CH(CH3)2

H

COOH

Treatment with the indicated reagents yielded peptides with the indicated composition:

Cyanogen bromide: CB-1: Ala, Ile, Val CB-2: Arg, Gly, Leu, Lys, Met, Phe, Ser Chymotrypsin: CT-1: Leu, Lys, Phe CT-2: Ala, Arg, Gly, Ile, Met, Ser, Val Trypsin: T-1: Leu, Lys T-2: Arg, Phe, Ser T-3: Ala, Gly, Ile, Met, Val

N-terminal analysis of peptide CB-1 revealed isoleucine. Carboxypeptidase treatment of the peptide liberated a nine-amino acid peptide and alanine.

What is the sequence of this peptide? 9. A peptide containing 10 amino acids was analyzed and found to contain the following

amino acids after complete hydrolysis: Ala Arg Asp Gly Ile Leu Lys Met Phe Ser Treatment of the intact peptide with phenylisothiocyanate in base, followed by anhydrous

HF and mild acid, yielded the following compound and a 9-amino acid peptide.

N

CCH2

NHC

S

O Treatment with the indicated reagents yielded peptides with the indicated composition Cyanogen bromide: CB-1: Ala, Gly, Leu, Lys, Met, Phe, Ser CB-2: Arg, Asp, Ile Chymotrypsin: CT-1: Gly, Leu, Phe CT-2: Ala, Arg, Asp, Ile, Lys, Met, Ser Trypsin: T-1: Gly, Leu, Lys, Phe, Ser T-2: Ala, Arg, Met T-3: Asp, Ile Treatment of the intact peptide with LiBH4 followed by acid hydrolysis liberated a 9-

amino acid peptide and the following compound.

CH

CH2OH

H2C CH2OH

+H3N What is the amino acid sequence of this peptide? [Use 3-letter code]

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10. The following is the reaction of dansyl chloride with a polypeptide chain:

NCH3 CH3

S

Cl

H2N C

H

R

C

NCH3 CH3

S OO

HN C

H

OO

O

R

C

ODansyl Chloride

1-Dimethylaminonapthalene-5-sulfonyl chloride

+Base

HCl

H+

(a) What is the purpose of the H+–catalyzed step? (b) Why is this reagent better than Sanger’s reagent?

Secondary Structure of Proteins

1. Secondary structure describes the three-dimensional shape assumed by which atoms in a

polypeptide chain? What bonds/interactions do these atoms form in secondary structures? 2. Briefly indicate what is meant by torsion angles and how different amino acid side chains

affect such angles. 3. Draw a peptide bond and indicate the atoms that lie in the plane. Indicate why these atoms

lie in a plane. Briefly discuss how this planarity affects protein 3-dimensional structure.

4. Diagram the α-helix (protein secondary structure) and indicate its important structural features.

5. Show how two regions of a polypeptide chain are held together to form an antiparallel β-pleated sheet (you need only draw as much structure as is necessary to show two bonds holding the regions together). What characteristics distinguish this form of secondary

6. Show how a second region of polypeptide chain would interact with the following in an anti-parallel β-sheet and indicate the important features of this form of secondary structure.

H

CN

C

O

RO

CN

HH 7. Show how a second region of polypeptide chain would interact with the following in a

parallel β-sheet. H

CN

C

O

RO

CN

HH 8. Provide a diagram illustrating the super secondary structures β-Meander and β−α−β.

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9. Collagen, a fibrous protein of connective tissue, is a triple helical cable – i.e. three protein components are wound around one-another. Collagen has 33% glycines residues. Explain why this is so. Be specific. Suggest how the glycine residues might be arranged along the polypeptide chain. Justify your answer.

10. Draw a peptide bond and indicate the atoms which lie in the plane. Indicate why these atoms lie in a plane. Briefly discuss how this planarity affects protein 3-dimensional structure. Your answer must include comments describing the usual structural relationship between the α-carbon atoms of adjacent amino acids, constraints on the orientation of amino acid side-chains, and identity of bonds and constraints to free rotation defined by Ramachandran as limiting possible secondary structures.

Tertiary and Quaternary Structure of Proteins

1. Identify two methods commonly used to determine the three-dimensional structure of

proteins. For each method, identify the major limitation that prevents application of the method to all proteins.

2. What are the advantages and disadvantages of electron diffraction and NMR in determining the three-dimensional structure of a protein?

3. Briefly discuss the role of hydrogen bonding in protein 3-dimensional structure. Identify the level of structure being discussed and provide an example of the atoms involved in forming the relevant H-bonds.

4. Illustrate using specific structures how water can form hydrogen bonds with itself or with an amino acid side chain. Briefly discuss how each of these hydrogen bonds contributes to protein three-dimensional structure.

5. Illustrate what is meant by an ion-polar bond using the side chains of glutamic acid and serine. Briefly discuss the importance of such bonds in determining the 3-dimensional structure of a protein.

6. List two ways different polypeptides are held together, illustrating one of the ways with the amino acid cysteine.

7. In the space to the right of each of the following orders of protein structure write the letter(s) of the description(s) which apply to it. Primary: ______________________ Secondary: _____________________ Tertiary: ______________________ Quaternary: ____________________

a. Involves non-covalent interactions. b. Has a specific pattern for each protein under physiological conditions. c. Always involves covalent bonds. d. Dictates the other orders of structure. e. Involves more than one polypeptide. f. Is random and has no particular pattern in a given protein. g. Involves hydrogen bonds only between atoms of the polypeptide backbone. h. Involves electrostatic and hydrophobic interactions between side chains. i. May involve disulfide bonds.

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8. Proteins are said to exhibit conformational flexibility. Briefly describe what is meant by this term and the features of the bond(s) which determine a protein’s conformation which allow proteins to exhibity such flexibility.

9. Briefly distinguish between van der Waals interactions and hydrophobic forces and indicate which is most important in causing such molecules to be located in the interior of proteins.

10. Briefly summarize the observation that suggests that the three-dimensional structure of a protein is determined by its primary structure (amino acid sequence).

11. What two proteins or processes assist in the folding process giving rise to a protein’s functional three-dimensional structure?

12. What are chaperones, what do they do, and how do they do it (general terms, no mechanistic details).

13. Briefly indicate the role of protein disulfide isomerase in protein folding.