Exam 2 Review Questions - Harvard Universitysites.fas.harvard.edu/~lsci1a/Review2Prob.pdf · 2009....

LS1a Midterm Exam 2 Review Session Problems

1. [Membranes] The bacteria Pseudomonas putida can alter the fluidity of its

lipid membrane using an enzyme called cis-trans isomerase, which catalyzes the reaction below on molecules that make up the lipid membrane:

O

HO

Pseudomonas putida

cis-transisomerase

A

B

O

HO

(straight chain)

(kinked)

a. Identify the cis- and trans- double bonds in the diagram above.

b. Which of the above geometric isomers (A or B) would increase the fluidity of a cell membrane? Briefly explain your answer.

Life Science 1A – Fall 2006 Exam Review 2 Questions

2. [Membranes] Phosphatidylcholine is a phospholipid that contains a pair of fatty acid chains. The following three forms of phosphatidylcholine (PC#1, #2, and #3) contain different pairs of identical fatty acid chains. The number of carbons and double bonds in the fatty acid chains contained in each PC are shown below.

Number of carbons Number of cis double bonds per fatty acid chain per fatty acid chain PC#1 22 0 PC#2 18 1 PC#3 18 0

Rank the three forms of phosphatidylcholine in terms of their likely melting temperature (Tm): (12 points)

Highest Tm ______ > ______ >______ Lowest Tm


3. [HIV and membrane fusion] T-20, an inhibitor of HIV membrane fusion, is a polypeptide fragment of the C-terminal α-helix of HIV gp41.

a. How does treatment of patients with T-20 inhibit the infection of host cells

with HIV?

b. Other membrane fusion inhibitors are being developed that are fragments

of the N-terminal helix of gp41. How would treatment of patients with an N-terminal helix fragment prevent membrane fusion?

c. If T-20 and the new N-terminal helix fragment were mixed together before being administered to HIV patients, would you expect this “combination therapy” to be more effective than either drug alone? Explain.


4. [Ion channels and membrane potential] In animal cells, the internal concentration of Na+ (15 mM) is much lower than the external concentration (145 mM), and the internal concentration of K+ (140 mM) is much higher than the external concentration (5 mM). To maintain this energetically unfavorable situation, cells employ ion pumps to actively transport Na+ and K+ against their concentration gradients. The Na+-K+ ATPase is an ion pump that is present in the plasma membrane of most animal cells. It uses the energy of ATP hydrolysis to pump Na+ out of the cell and K+ into the cell.

a. The Na+-K+ ATPase pumps 3 Na+ ions out of the cell, and 2 K+ ions into

the cell per ATP consumed. Please explain how the function of this pump may contribute to the formation of an electrochemical (charge) gradient across the plasma membrane.

b. Membrane proteins are often insoluble in solution (they won’t dissolve in water). Based on their chemical properties, please explain why this is.

c. To solubilize membrane proteins, biochemists often add detergents to isolated animal cell membranes. Detergents are small amphipathic molecules with a hydrophobic and a hydrophilic end. How would detergents help make membrane proteins more soluble in aqueous (water) solution?


d. You decide to purify and study the Na+-K+ ATPase. To do so, you first solubilize the Na+-K+ ATPase out of the plasma membrane using detergents. Then you isolate your protein and slowly replace the detergent molecules with new phospholipids to reconstitute a phospholipid bilayer. In this manner you can recreate phospholipid bilayers where the only protein component is your Na+-K+ ATPase. If you do this experiment in a solution containing Na+, K+ and ATP, you reconstitute phospholipids vesicles with the chemical characteristics shown below, where the concentration of all Na+, K+ and ATP are equal on both sides of the reconstituted plasma membranes.

Notes: The total amount of aqueous solution inside the vesicles can be assumed to equal to the amount of aqueous solution outside of the vesicles. The diagram is an artistic rendition and not draw to scale.

Over time you observe that the concentration of ATP inside and outside of the vesicles drops to almost 0 (is converted to ADP and Pi), however the concentrations of Na+ and K+ inside and outside of the vesicles is unchanged. Please explain how this could have occurred.

Na+ [150 mM]

K+ [150 mM]

ATP [20 mM ]

Na+ [150 mM]

K+ [150 mM]

ATP [20 mM ]

Na+ [150 mM]

K+ [150 mM]

ATP [20 mM]

Na+ [150 mM]

K+ [150 mM]

ATP [20 mM]


5. [Secretion] In order to investigate the subcellular and viral locations of various HIV proteins, you fuse GFP to different HIV proteins and observe their locations using a microscope.

For the following proteins, indicate whether you would observe GFP fluorescence in the ER, Golgi, plasma membrane, cytosol, and/or newly budded viruses. If possible, indicate which side of the compartment(s) fluorescence will be observed (lumenal/extracellular vs. cellular).

a. GAG

b. gp120

c. gp41 (assume GFP on extracellular part of protein)

d. A colleague of yours attempts the same experiment, but gets different

results for gp41. They find that all of the GFP fluorescence is always located in the cytosol. You look at their gp41 fusion protein construct and notice that they have made constructs that introduce GFP at the N-terminus. Based on what you know about signal sequences explain how their approach could have led to this difference.


6. [Secretion] The toxin produced by the bacterium Clostridium botulinum is a potent neurotoxin. When used in high doses, the toxin causes death through paralysis. However at low doses the toxin can be used for cosmetic purposes or to treat overactive muscles.

Botulinum Toxin (BTX) prevents neurons from being able to activate muscle contractions. BTX accomplishes this through proteolytic activity (by degrading specific proteins).

a. One explanation is that BTX prevents the budding of neurotransmitter

containing vesicles from the Golgi Apparatus. If this explanation is correct, what class of proteins would BTX most likely target for degradation? Briefly explain.

b. Another explanation is that BTX prevents the fusion of neurotransmitter containing vesicles with the plasma membrane. If this explanation is correct, what class of proteins would BTX most likely target for degradation? Briefly explain.


c. To determine which explanation is more probable, the trafficking of a membrane protein is investigated. In the absence of BTX, in what different cellular compartments could the membrane protein be found? Please indicate on the diagram below.

d. In the presence of BTX, the membrane protein is found in intracellular vesicles, however none is seen on the plasma membrane surface. Which explanation, a or b, does this data support? Explain.


7. [Transcription and HIV] You would like to identify targets for a new antiviral agent that can selectively inactivate HIV proteins. Based on your knowledge of HIV biology from LS 1a, you decide to take two different approaches.

a. In one approach you seek to reduce the transcription of HIV genes. Which

HIV protein is a logical target based on the effect it has on the activity of RNA polymerase?

b. Would the inactivation of this protein be expected to disrupt the transcription of host cell genes? Why or why not? Provide a concise answer in four or fewer sentences.

c. In a second approach you seek to disrupt the translation of HIV proteins. Which HIV protein is a logical target based on the effect it has on which HIV mRNAs are translated?

d. When this protein is inactivated, the translation of most HIV proteins is significantly decreased. However, the translation of some HIV proteins are not decreased. Why? Provide a concise answer in four or fewer sentences.


e. One of your colleagues suggests that another suitable target might be the protein(s) responsible for processing (5’-capping and 3’-polyadenylating) HIV pre-mRNA transcripts. Is this a good idea? Why or why not? Provide a concise answer in four or fewer sentences.


8. [HIV] Consider three strains of HIV. One is wildtype HIV, mutant 1 has a point mutation in the TAR sequence, and mutant 2 has a deletion mutation in the sequence encoding TAT.

Three different reporter genes (green, yellow, and cyan fluorescent proteins) were inserted into each of these three viral genomes as shown below.

a. What is the function of TAT and to what must it bind to fulfill its function?

To study the effects of the mutations, you infect cells with these strains of HIV and measure the fluorescent intensity over 24 hours. Use your results shown in the following graph to answer parts b-d.

TATTAR Green Fluorescent Proteinwildtype

X

X

mutant 2 TATTAR Cyan Fluorescent Protein

mutant 1 TATTAR Yellow Fluorescent Protein


b. You infect cells with the wildtype strain and observe the above results. Explain.

c. You infect cells with the tar point mutant, mutant 1 and observe the above results. Explain.

d. You infect cells with the tat deletion mutant, mutant 2 and observe the above results. Explain.


e. You co-infect cells with the wildtype strain and mutant 1 and observe the following results. Explain.

f. You co-infect cells with the wildtype strain and mutant 2 and observe the following results. The graphs for both strains are identical. Explain.


g. You co-infect cells with mutant 1 and mutant 2. Graph your predicted results and explain.

9. [Amino Acid Reminder and Translation] Aminoacyl-tRNA synthetases attach specific amino acids to their appropriate tRNAs in preparation for protein synthesis. The synthetase that attaches valine to tRNAVal must be able to discriminate valine from threonine, which differ very slightly in structure.

a. Please draw the structures of valine and threonine below. How do their

structures differ?


b. In the synthesis site, how is discrimination between amino acids generally accomplished?

c. In the editing site how is discrimination generally accomplished?

d. To bind to valine the binding pocket of the synthesis site is lined with hydrophobic residues. Such a binding site permits valine to bind well, but does not fully exclude threonine. Threonine has a similar shape but does not bind as well as valine, why?

e. The editing site is much more specific for threonine. It has an appropriately positioned residue that can interact with threonine and form a specific type of bond with threonine, but not valine. Based on the structures of threonine and valine, what type of bond is most likely formed?


10. [Translation] Tay-Sachs disease is an inherited disorder that results from the inactivation of the lysosomal enzyme β-N-acetylhexosaminidase A. The inactivation of this key metabolic enzyme results in the swelling of neuronal cells.

The most common form of the disease results from the incorporation of a four-base pair insertion into the middle of the gene:

Normal Gene: ATATCCTATGGCCCTGATAGCC...

Altered Gene: ATATCTATCCTATGGCCCTGATA...

Properframe

Four-baseinsertion

a. Why might this insertion result in a dramatic loss in enzymatic activity?

Explain in 2-3 concise sentences.

b. Later biological studies on the mutant protein indicated that the protein is

much smaller than the natural protein.

Using the sequences provided, provide a concise explanation.

Exam 2 Review Questions - Harvard Universitysites.fas.harvard.edu/~lsci1a/Review2Prob.pdf · 2009....

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