Question 3. A new startup company hires you to help with their product developme

Question

Question 3. A new startup company hires you to help with their product development. Your task is to find a protein that interacts with a polysaccharide.

a) You find a large protein that has a single binding site for the polysaccharide cellulose. Which amino acids might you expect to find in the binding pocket of the protein? What is the strongest type of interaction possible between these amino acids and the cellulose?

In the course of your research you discover a small polypeptide that is associated with cellulose. You want to design an enzyme that will bind this small polypeptide. You design the enzyme shown below.

b) In the table below please indicate which amino acid you placed in each position to give the strongest possible interaction between your enzyme and the small polypeptide. For instance, you would choose an amino acid for position D that most strongly interacts with the carboxyl group of the small polypeptide. Also list what interaction occurs at each position (choose from: covalent bond, ionic bond, hydrogen bond, or van der Waals forces).

Amino Acid position Amino Acid Interaction

A   

B

C

D

E

F

Question 4. Prions (pronounced pree-ahns) are agents that cause a novel type of fatal brain disease. Bovine spongiform encephalopathy (BSE or mad cow disease), sheep scrapie and Creutzfeldt-Jakob disease (CJD) of humans are examples of prion diseases. Prions enter cells and convert normal proteins found within the cells into prions just like themselves. The normal cell proteins have all the same "parts" as the prions--specifically the same amino acid building blocks--but they fold differently.

a) What is the primary structure of a protein? What force or forces (covalent bonds, ionic bonds, hydrogen bonds, or van der Waals forces) are involved in primary structure?

b) What is the secondary structure of a protein? What force or forces (covalent bonds, ionic bonds, hydrogen bonds, or van der waals forces) are involved in secondary structure?

c) What is the tertiary structure of a protein? What force or forces (covalent bonds, ionic bonds, hydrogen bonds, or van der waals forces) are involved in tertiary structure?

d) Do you expect both the normal and the infectious versions of a prion protein to have the same primary structure? Why or why not.

e) Do you expect both the normal and the infectious versions of a prion protein to have the same tertiary structure? Why or why not.

Question 6. For each of these methods of separating proteins, describe the principle of the method, namely what property of proteins allows their separation by this technique

a) ion-exchange chromatography;

b) size-exclusion chromatography.

Question 7. If two polypeptides have the same molar mass, name two methods you could employ to separate them. What is the principle of the method of separation?

Question 8. How is beta- Mercaptoethanol used in establishing the amino acid sequence of polypeptides? Why is it used to prepare the sample for gel-electrophoresis?

Question 9. How would you make 100mL of a 0.05M phosphate buffer at pH 7.5 using:

Na2HPO4*7H2O

NaH2PO4*1H2O

Question 11. The charge of a molecule in solution is determined by what?

Question 12. What are the two general types of anion exchange chromatography (IEC)?

Question 13. Use IEC to purify these AA:

Aspartic acid

Phenylalanine

Leucine

Alanine

Lysine

What resin will you use? What pH of the buffer? What is the order of elution?

Question 14. When does absorbance occur (generally)?

Question 15. Biological molecules absorb energy in which regions of the electromagnetic spectrum?

Question 16. What is “salting in”? “Salting out”? Explain the mode of action of ammonium sulfate in the context that you are familiar with thru AP purification.

Question 19. Explain the procedure of western blotting. Draw a schematics of WB sandwich, indicate poles, and explain what happens if the poles were reversed

Question 20. You have partially purified an enzyme using non-denaturing conditions. The preparation still has several bands when analyzed by SDS gel electrophoresis (Figure, lane 1).You want to identify the band(s) that are possibly the protein of interest and to completely purify the protein. You further analyze one portion of this starting material by gel filtration chromatography and the enzyme activity eludes as a single peak of activity with a molecular mass of approximately 300 kDa. You pool these fractions and determine the specific activity and analyze the protein sample by SDS gel electrophoresis (Table and Figure, lane 2). You analyze another portion of this starting material by IEC and the majority of the enzyme activity is found in a single fraction. You determine the specific activity of this fraction and analyze the protein sample by SDS gel electrophoresis (Table and Figure, lane 3).

A. Identify the protein band(s) on the gel which are most likely to be responsible for the enzyme activity? (You can give approximate sizes or indicate directly on gel with arrows, asterisks, circles, etc.) Explain your answer.

B. What predictions, if any, can you make about subunit composition of the enzyme? Explain your answer.

C. How close to being completely purified will the enzyme be if the gel filtration and isoelectric focusing are carried out as sequential steps? In other words, how many contaminating bands and how much would you still expect in a preparation in which the two procedures were carried out sequentially (i.e., either subject the 300 kDa gel filtration peak to isoelectric focusing or subject the pH 6.9 isoelectric focusing fraction to gel filtration)? You can indicate on the figure the contaminating bands or draw a figure of the expected gel.

D. Assuming that the fold-purification and yields are similar if the steps are done sequentially as compared to being done separately, what would be the expected specific activity following combined gel filtration and isoelectric focusing?

Question 21. Complete a column “Yield” and a column “Purity” in a typical enzyme purificationsummary table is shown below. Refer to Appendix 3 for the explanations.

Explanation / Answer

1. Protein with single binding site would be glycine. the protein will have dispersion forces in them.

2. A : Valine : Van der waals

B : Serine : Hydrogen bonding

C : Glutaric acid : Hydrogen bonding

D : Asparagine : covalent bond

E : Phenylalanine : van der waals

F : Glutamic acid : ionic bond

4)

a) Primary strucure of protein is the actual covalently linked amino acids.

b) Secondary structure is interaction of groups in hydrogen bonding fashion. These are strng bonds and help fold the protein structure.

c) Tertiary structure is folding of proteins in order to have maximum van der waals forces between hydrophobic side groups and the hydrophilic ends are lying outside the cage.

d) normal version will differ from the infectious version as after infection the bonds would be weaker and protein would not be in folded position.

e) Infectious version here would have lower number of van der waals forces and normal version will have in turn greater number of forces due to symmetry and no outside additive.

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