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LS1a Fall 2014 Practice Problem Set #4 1. Phospholipids typically form bilayer structures when present in aqueous solutions. Briefly explain how introducing cis-double bonds into saturated fatty acid chains of a phospholipid will affect the melting temperature (Tm) and the fluidity of that membrane bilayer. 2. Certain molecules such as oxygen (O2) can freely diffuse across cell membranes. Which of the following molecules do you expect to freely diffuse across cell membranes (indicate by circling)? 3. Organisms can regulate their lipid composition in response to temperature. Fish are particularly rich in “Omega-3” and “Omega-6” fatty acids, which are unsaturated fatty acids containing cis-double bonds (the “omega- #” names refer to the location of the cis-double bond). On the other hand, poultry and mammalian meat sources (chicken, beef, etc) tend to be richer in saturated fats, which do not contain any double bonds. a) In light of what you know about plasma membranes, why is it not surprising that fish membranes are rich in unsaturated fats while poultry and mammalian membranes are rich in saturated fats? (Hint: Fish are cold-blooded, whereas birds and mammals are warm-blooded). Regulating membrane fluidity is especially important for organisms such as bacteria that cannot regulate their own temperature. b) Consider a colony of bacteria that suddenly undergoes a drastic drop in temperature. What consequence would this have on the fluidity of the membranes of the bacteria? 1 c) Some bacteria have enzymes that can adjust the length and saturation of the fatty acid chains. If this drop in temperature was gradual, what could the bacteria do to combat the change in membrane fluidity? d) If the bacteria were suddenly shifted to a very high temperature after gradually adjusting to a lower temperature, how would membrane permeability be affected? 4. In your studies of cells, you discover two new transmembrane proteins, A and B, which are present on the cell surface. To further investigate their distribution in the plasma membrane, you use fluorescent tags to label them. a) You first label Protein A with a fluorescent tag and examine its localization in the cell’s membrane using a microscope. You find it is distributed diffusely across the cell surface. You then use FRAP (“Fluorescence Recovery After Photobleaching”) to determine the mobility of protein A in the membrane. You notice that after bleaching an area of the cell membrane 90% of the fluorescence is recovered in this area within 5 minutes. Draw the recovery graph you would expect and briefly explain these results. b) Next you label protein B with a fluorescent tag and find it is also distributed diffusely across the cell surface. However the FRAP results are very different. After bleaching an isolated area of the cell membrane, only 25% of the fluorescence is recovered in this area within 5 minutes. Draw the recovery graph you would expect and briefly explain these results. 2 c) You further examine the mobility of protein A in cells that have a defect in cholesterol production. The membranes of these cells have very little cholesterol compared to the cells used above. If you repeat your FRAP experiments from part (a) using these cells, how do you predict the mobility of protein A would compare with your previous observation at both low and high temperatures? Briefly explain your answer. 5. Porins are transmembrane proteins with hollow centers through which small molecules can diffuse. The structure of one is shown below (in sideview). a) What is the predominant secondary structure in a porin? b) When the sequence of a single yellow protein strand is analyzed, it is discovered that consecutive amino acids alternate between hydrophobic and hydrophilic sidechains. Does this make sense? Briefly explain. 3 c) If this porin protein is tagged with a fluorescent molecule and FRAP is performed, the results below on the left are seen. If another transmembrane protein (Protein A) is tagged with a fluorescent molecule and FRAP is performed, the results below on the right are seen. Which of these proteins is more mobile in the plasma membrane? Briefly explain. d) Brief treatment with proteases (enzymes that catalyze the breaking of peptide bonds) cleaves some of peptide bonds that are on the surface of a protein and more accessible. If the membranes containing either tagged Porin or tagged Protein A are briefly treated with a protease and then FRAP is preformed, the mobility of both proteins are indistinguishable from the results shown above for Protein A. Based on this information, provide a plausible explanation for the mobility of Porin in the absence and presence of protease treatment. 6. An engineered ribozyme is a small type of nucleic acid that can catalyze a chemical reaction. Shown below is an example of a ribozyme (black RNA) along with its substrate (red RNA). This particular ribozyme can cleave the phosphodiester bonds of its substrate. The site where the ribozyme cleaves the substrate is indicated. G A CGCGG GCGCC GA UAG U C AGCUCG UCGAGC C A A U Cleavage C G site A U 5' 3' 4 3' 5' a) The ribozyme catalyzes the cleavage of the red substrate by forcing it to assume a conformation that increases the likelihood of a so-called transesterification reaction in which the 3’-5’ phosphodiester bond at the cleavage site between two adjacent ribonucleotides within the substrate is cut in an intramolecular reaction where the 2’ OH group attacks the phosphodiester backbone. Using the drawing shown below, draw in the products of this reaction. b) Because RNA is difficult to work with, scientists sometimes attempt to make ribozymes out of DNA. If you made the entire ribozyme (black portion) out of DNA and it adopted the same shape, would it be able to cleave the substrate? Briefly explain. c) If the ribozyme (the black portion) were still made of RNA, but the substrate (the red portion) was made of DNA, could the ribozyme still catalyze the cleavage of the substrate’s phosphodiester bond? 5 Answers 1) Introducing fatty acids with cis-double bonds will increase the space between adjacent fatty acid chains and diminish the van der Waal’s forces that exist between nearby chains, thereby lowering the melting temperature and making the membrane more fluid. 2) Small uncharged, polar molecules are permeable to the membrane. 3a) In order for membranes to function properly they need to maintain a certain fluidity. The fluidity of a membrane depends both on temperature and on its composition. One component that can be varied in membrane composition is the degree of saturation of the fatty acid tails. Fewer cis-double bonds will allow more van der Waals interactions between the fatty acid tails, and the membrane will have a higher Tm. Since fish are cold-blooded, their cellular temperature depends on their environment. If they are living in relatively cooler waters (compared to the average mammal’s body temperature) it is not surprising that their membranes would be rich in unsaturated fats to decrease the van der Waal’s interactions and subsequently decrease their plasma membrane Tm. Thus their membranes resist becoming more solid-like at this cooler temperature. 3b) The fluidity of the membranes would decrease as they would become more solid-like. 3c) In response to cold the bacteria could shorten their fatty acids chains, synthesize more fatty acids with cis-double bonds, or increase the concentration of cholesterol in the membrane. Each of these processes would reduce phospholipid packing and therefore the van der Waal’s interactions between the fatty acids, increasing the fluidity of the membrane. 3d) Membranes would become too fluid which could cause them to become “leaky” and allow ions to cross. 4a) Photobleach 100 90 percent fluorescence time (min) 6 5 The rate of recovery of fluorescence in the bleached area is a measure of the rate of the mobility (lateral diffusion) of Protein A. Protein A is mobile in the lipid bilayer so after bleaching the non-fluorescent Protein A moves out of the area and fluorescent Protein A diffuses in. Since some of the proteins will have permanently lost their fluorescence the recovery is not 100%. 4b) 100 percent fluorescence 20 time (min) 5 Protein B has limited mobility in the lipid bilayer so after bleaching much less of the fluorescence is recovered after 5 minutes. The majority of the bleached Protein B is relatively immobile; it might be anchored to proteins inside the cell. Therefore, bleached Protein B molecules are unable to diffuse out and be replaced by other fluorescent Protein Bs. 4c) At high temperatures cholesterol decreases membrane fluidity. Cholesterol is present in high concentrations in the membranes of eukaryotic cells. If cholesterol was depleted from membranes of otherwise similar composition, it is likely that the absence of cholesterol would cause the mobility of protein A to be greater than what was previously observed. AND At low temperatures cholesterol increases membrane fluidity. If cholesterol was depleted from membranes of otherwise similar composition, it is likely that the absence of cholesterol would cause the mobility of protein A to be less than what was previously observed. 5a) Beta sheets 5b) Yes, the side chains in a beta sheet alternate between above the plane of the sheet and below the plane of the sheet. For porin the hydrophobic side chains are in contact with the hydrophobic fatty acid tails of the membrane phospholipids and the hydrophilic side chains point into the aqueous environment of the channel. 5c) Protein A is more mobile. The fluorescence in the bleached area of the membrane for Protein A recovers fairly quickly whereas there is virtually no recovery of fluorescence in the bleached area of the membrane where porin is labeled. 7 5d) Porin could be anchored to another protein restricting its mobility in the membrane. When the membranes are treated with protease, it breaks the association between porin and the fixed protein and allows protein to be mobile in the membrane. 6a) 6b) Yes. So long as the DNA is able to contort the RNA into the correct conformation, it will accelerate the reaction. 6c) No. The 2’ hydroxyl, which is present in RNA but not DNA, has to be in the substrate for this reaction to occur. 8