7.06_2004_PS1 - 7.06 Spring 2004 PS 1 1 of 6 7.06 Problem...

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Unformatted text preview: 7.06 Spring 2004 PS 1 1 of 6 7.06 Problem Set 1 1. You would like to purify a protein(s) that is responsible for some activity (call it X) in the cell. Fortunately, you have at your disposal a very simple colorimetric assay to monitor the activity you are interested in. a) List at least 3 possible techniques you could use to purify the activity out of a crude cell lysate and the basis by which these methods separate molecules of interest (note: you do not know the identity of the activity producing molecule, so antibodies cannot be used). How do you know you have isolated the activity of interest? b) You use the colorimetric assay to measure the enzyme’s activity in the crude lysate and found it to be 5000 units. When you examine your fractions, however, none seem to contain any measurable activity. How could this have happened? c) As previously mentioned, your lysate contained 5000 units of activity. But, unlike the situation outlined in (b), what if one fraction contains 50, 000 units of activity. Under what circumstances could this happen (i.e. how could you have 10x more activity in your purified fraction than in your total lysate)? d) You decide to use Gel Filtration to purify your activity. What property are you exploiting by using gel filtration? How would you actually measure this property? 7.06 Spring 2004 PS 1 2 of 6 e) You perform the gel filtration experiment and measure the activity using your nifty colorimetric assay. The following results are obtained (the elution position of molecular weight standards is superimposed on the profile). Elution Profile 10 kDa 30 kDa 100 kDa 70 kDa 50 kDa 140 120 Activity (U) 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 90 Fraction Number What is the estimated molecular mass of the molecules in the sample with the highest activity? 100 7.06 Spring 2004 PS 1 3 of 6 f) However, after performing SDS-PAGE on fractions containing the highest levels of activity and staining the gel with Coomassie Blue (a dye that reveals proteins), you observe the following: Fractions 49 MW Standards 50 51 52 100 80 70 50 30 10 How could you reconcile the difference between the molecular mass obtained by gel filtration with the one determined by SDS-PAGE? What if, instead of the gel shown in (f), you obtained the following pattern? Fractions MW Standards 49 50 51 52 100 80 70 50 30 10 What would you conclude about the protein’s organization given this gel? After successfully purifying the protein, you intend to determine its identity. List two methods that you could use to identify the protein of interest if you are given a test tube of purified enzyme. 2. You have decided to do a UROP and end up working in a lab that studies transport proteins. This doesn’t concern you, however, since you learned all about transport proteins in 7.06. Your graduate student advisor is attempting to create an in vitro system involving cellular Ca2+ flux in order to study Ca2+ triggered intracellular signaling. However, your advisor is busy with other problems and doesn’t have time to choose the 7.06 Spring 2004 PS 1 4 of 6 best Ca2+ pump for the system. He thus asks you to find the best Ca2+ pump for his system. (a) Being the expert that you are, you know the first thing to do is to collect different kinds of Ca2+ pumps to test. List two methods of enriching for Ca2+ pumps in the membrane. (b) Now that you’ve obtained the initial rates of Ca2+ intake by the pumps, you can determine the Vmax and Km of each Ca2+ pump. What does Vmax and Km tell you about a given Ca2+ pump? (c) Using the initial rate data below, calculate the Vmax and Km for each Ca2+ pump. 80 Ca2+ pump A Ca2+ pump B Ca2+ pump C Initial rate of Ca2+ export into liposome, Vo 70 Ca2+ pump D 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 2+ External [Ca ] uM (d) Based on this data, which Ca2+pump would you recommend to your graduate student and why? Also, why would some do some Ca2+ pumps have higher Kms than others? (e) Ca2+ pumps fall under the category of ATP-powered pumps. Explain how ATPpowered pumps work. Also explain how ion channels work, what the three types of transporters are and how each one works. 3. (a) It is known that capsaicin, the active ingredient in hot and spicy foods, increases the permeability of sensory neuron plasma membranes to cations. You speculate that there is a specific ion channel within these neurons that binds capsaicin and is responsible for this increased permeability. In your lab, you have the ability to visualize the calcium 7.06 Spring 2004 PS 1 5 of 6 uptake of cells using fluorescence microscopy and a calcium sensitive dye. How would you identify the capsaicin receptor? (b) You successfully clone the capsaicin receptor and decide to study its properties as an ion-channel using patch clamping. (i) The resting membrane potential of an oocyte not treated with capsaicin is –65 milliVolts. The sodium concentration of the patch electrode is 90 mM, and you have measured the intracellular sodium concentration to be 8mM. What is the ∆G for the inward movement of sodium ions at 37˚C? Is the process thermodynamically favored? (ii) When performing a single channel patch clamp of an oocyte expressing the receptor, you add 1µM capsaicin to the media and measure a current of 3pA. How many sodium ions does the capsaicin receptor let into the cell per second? (iii) You find the transport of cations by the receptor to be independent of ATP. What type of transport mechanism does the receptor use? (iv) How does the type of transport utilized by the capsaicin receptor differ from the transport mechansim of the Na+/K+ ATPase? 4. While studying families that have a severe skin blistering disease you find linkage to a gene you name epidermo-1. The genomic structure is shown below, exons are depicted as boxes, introns as straight lines: 1 2 3 4 Sequencing of affected individuals reveals a deletion of exon 3. You decide to try to develop a mouse model of the disease. (a) Using only a computer how would you determine whether mice have a homologous gene? and whether it has been shown to be expressed in the skin of mice? (b) You decide to recreate in mice the mutation found in the affected individuals. Based on the human disease phenotype your supervisor thinks that Epidermo-1 may be involved in forming cell-cell junctions and therefore may be essential during development of the mouse. How would you modify your gene targeting strategy so as to ensure the targeted mice were not embryonic lethal? (c) The mutant mice you generate develop a skin blistering disease. To try and investigate the function of Epidermo-1 you take skin sections from both wild type and mutant mice and using an antibody against the N-terminus you carry out some immunostaining. In 7.06 Spring 2004 PS 1 6 of 6 wild-type skin Epidermo-1 is found to co-localize with desmocollin and desmoglein, two proteins found in desmosomes. Since a portion of exon 3 is composed of a stretch of 22 hydrophobic amino acids that can form an a -helix, where might you expect to find the mutant Epidermo-1 protein? Why? (d) You attempt to culture keratinocytes from these mutant mice and find that, unlike wild type keratinocytes, they don’t form a compact monolayer and detach from the cell culture dish very easily. In an attempt to demonstrate that Epidermo-1 is involved in cellcell interactions at desmosomes you treat wild type keratinocytes with EGTA, a calcium chelator, and find that they detach from the dish just like the mutant cultures. Why does this occur? 5. Below are five of the ways by which proteins can associate with the plasma membrane: (i) as a single-pass transmembrane protein (ii) as a multi-pass transmembrane protein (iii) via a covalent fatty acid chain (iv) via an oligosaccharide to phosphatidylinositol (v) as a peripheral protein via electrostatic interactions or a lipid binding domain (a) Give an example of each and detail the conditions by which you could purify each of these proteins from the cell membrane. (b) Why is it that polypeptides that enter the bilayer tend to pass completely through it rather than changing direction midway? (c) You have cloned a multipass transmembrane receptor and are trying to determine whether the N-terminus is on the cytoplasmic or the extracellular side of the bilayer. How would the following information help you determine this: (i) position of glycosylated residues (ii) a series of monoclonal antibodies whose epitopes have been mapped (iii) a recombinant form of the protein that has been tagged at the N-terminus You find that the protein has four cysteine residues, two on the extracellular side, two intracellularly. Which are more likely to form disulfide bonds and why? ...
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