Unit2A Problem Set (1)

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© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 1 of 11 Unit 2, Part A, Problem Set – By Topic Based on material from Chapter 2 of the textbook: https://open.oregonstate.education/cellbiology/chapter/biological-membranes/ Topic 2.1 – The Chemical Features of Biological Membranes Learning Goals CLO refers to the course-level learning outcome, as described in the course syllabus. • List the four primary features of a biological membrane and explain why they are important for cellular function. CLO2, CLO3 • Explain how the chemical structure of a membrane (including lipids, carbohydrates and proteins) contributes to its function (as identified by the primary features listed above). CLO3 • Explain how the hydrophobic effect holds membranes together, and selectively excludes some molecules but not others. CLO2, CLO3 • Define the term ‘membrane fluidity’ in two different ways. CLO1 Topic 2.1 - Content Review Questions (i.e., Study group discussion points) 1. For the following molecules, examine the molecular structures and label the following regions (note that they may not all exist in each of the molecules: phosphatidylcholine, cholesterol, generic glycolipid, sodium dodecyl sulfate [SDS]): a. the polar region (differentiate between charged and uncharged molecules of the polar region) and the nonpolar region b. a region that would be stiff and inflexible c. a glycerol residue (Some molecules have a serine residue instead; does yours?) d. a region that could easily have C=C bonds added (How would that affect the structure of that region?) 2. For the molecules you found in Question 1, identify the lipids that would be able to form lipid bilayers on their own. Use the details of the structures that you drew to explain why or why not. 3. Define the four levels of protein folding and explain how each one is stabilized. 4. Self-assembly of macromolecules is an important concept. What do you think that means? 5. Are disulfide bridges covalent or noncovalent interactions? How do they form? Why are they considered to be uncommon in the cytosol? 6. Find images of all 20 amino acids (don’t memorize them!) online. Critically assess the structures and identify the following: a. The portion of the amino acid that is common to all of them. b. The portion that is unique to each amino acid, known as the R group or side chain.

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 2 of 11 c. The functional groups that will form the peptide bond. Will anything be lost/gained during that reaction? How do these parts relate to the “N” and “C” terminus of a protein? d. The next questions are specifically for the R groups. Based solely on their structure, identify the following: ▪ Any R groups that you would expect to have acidic properties. Will they gain or lose a proton during that reaction? ▪ Any R groups that you would expect to have basic properties. Will they gain or lose a proton during that reaction? ▪ Any R groups that would not be able to form H-bonds with water. ▪ R groups that would form H-bonds but would not be acidic or basic. ▪ R groups that could interact ionically with their neighbors. ▪ Any R groups you would consider to be “big” or “small” relative to the others.

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 3 of 11 Topic 2.1 - Practice Problems (Data analysis & problem solving. More likely to be exam-style) Problem 2.1.1 (Walkthrough Available) The experiment described here is of historical importance as it provided the first decisive evidence that the folding of proteins was dependent only on the primary structure of the protein and that it was a result of molecular self-assembly (i.e. based on chemical properties). A Nobel prize was awarded for this work way back in 1972. Background: This experiment deals with the structure and activity of ribonuclease, an enzyme that degrades RNA. It is produced in the pancreas. It is one of the first proteins for which the amino acid sequence was known. The protein's enzyme activity occurs only when the molecule is properly folded. As the molecule denatures, the enzymatic activity is lost. The molecule has 4 disulfide bonds (C- S-S -H) that hold the chain together (see picture on the right). These form between cysteine residues. If the molecule is to be completely denatured, these bonds must be broken. Disulfide bridges are broken by placing the protein in a solution of 8 M urea and beta mercaptoethanol (HS-CH 2 -CH 2 -OH, reducing agent). The urea interferes with formation of hydrogen bonds and the reducing agent provides a reducing environment, in which the disulfide bond will break, leaving 2 – SH groups (one on each cysteine). The plot on the right (top) shows how the enzymatic activity decreases as the mean number of disulfide bridges per ribonuclease molecule decreases during denaturation. Each disulfide bridge produces two SH groups: -C-S-S-C- → C-S H H S-C- (oxidized) (reduced) If the reducing agent and the urea are removed the protein will slowly oxidize in the presence of air. The S-S bonds will reform and enzyme activity is restored, as shown in the lower graph on the right (Figure 2). a) Describe how enzyme activity changes as the mean number of disulfide bridges decreases. Why do you think the enzyme activity is affected this way? b) Compare the changes in enzyme activity and reformation of S-S groups change as reoxidization progresses. What does this tell you? c) What are some of the assumptions we are making in this experiment? d) Why do you think there is a lag between the formation of S-S bonds and the restoration of enzyme activity? e) Based on the data been presented here, what did this landmark research contribute to our understanding of protein folding? (Source of data: Anfinsen et al. 1961 PNAS 47, 1309, and White 1960 J. Biol. Chemistry 235, 383)

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© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 4 of 11 Problem 2.1.2 The structure and chemical properties of a lipid bilayer is determined by the particular components of its lipid molecules. As a thought experiment, think about what would happen if: a) Phospholipids had only one hydrocarbon tail instead of two? b) The hydrocarbon tails were much shorter than normal, say, about 10 carbon atoms or less? c) All of the hydrocarbon chains were saturated? d) Would you expect the permeability of a synthetic membrane, made entirely of phospholipids, to be greater or less than that of a ‘real’ membrane? Why? e) Would you expect a single-celled, freshwater organism have a membrane that is more or less permeable than a single-celled marine organism? Why? Problem 2.1.3 Here are partial amino acid sequences of normal human hemoglobin and three variants (mutants) that occur normally in the human population. Some of these hemoglobin molecules lead to defective function of the molecules while others function quite normally. a) Which version is most likely to result in a phenotypically "silent" mutation? Why? b) Which version is likely to result in the largest disruption of the three-dimensional structure of the protein? Why? Problem 2.1.4 Explain why it would be incorrect to say the following: a) Lipids in a lipid bilayer are immobile. b) Lipids in a lipid bilayer rapidly flip from one leaflet to the other. c) When a protein is made of more than one polypeptide, disulfide bridges must be used to hold the two subunits together. d) Oxygen and water molecules cannot pass through a lipid bilayer unless there are proteins present. e) Lipid bilayers are assembled with the help of chaperone proteins. f) A lipid bilayer and a biological membrane are synonyms of each other. They define exactly the same thing.

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 5 of 11 Problem 2.1.5 Researchers wanted to examine whether the distribution of a phospholipid, known as phosphatidylserine (PS), was distributed symmetrically in healthy plasma membranes of red blood cells. Membranes were isolated, broken into fragments, and then left to reform spontaneously into vesicles. From this, two different populations of vesicles were identified: • Right-side-out vesicles (ROVs), in which the original extracellular leaflet of the plasma membrane faces the exterior (equivalent to the orientation of the intact plasma membrane); and • Inside-out vesicles (IOVs), in which the original extracellular leaflet faces the interior of the vesicles and the cytoplasmic leaflet faces outwards. A fluorescent marker for PS was used to label the leaflet of the membrane that was exposed to the outside in each population of vesicles (ROVs & IOVs). The images of this labeling are shown above. Note: to help you interpret these images, a dashed line has been added to outline each of the vesicles in the image. a) Describe what can be observed in each panel. b) What conclusions can you draw about the distribution of phosphatidylserine in the plasma membrane of the red blood cell? Explain your reasoning.

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 6 of 11 Topic 2.2 – Maintaining Fluidity in the Membrane Learning Goals CLO refers to the course-level learning outcome, as described in the course syllabus. • Identify the two primary structural features that are required to be considered a membrane lipid, and explain why cholesterol is considered a membrane lipid even though it does not have these features. CLO1, CLO3 • Describe how the lipid composition can influence membrane fluidity, and how membrane fluidity can be maintained under different environmental conditions, especially temperature and pressure. CLO2, CLO3 • Explain how Fluorescence Recovery After Photobleaching (FRAP) works, and interpret results of FRAP experiments. CLO4 Topic 2.2 - Content Review Questions (i.e., Study group discussion points) 1. What are the major differences between a synthetic phospholipid bilayer and a biological membrane? 2. How do cells adjust their membrane composition to maintain fluidity of their lipid bilayers in varying conditions? 3. Despite appearances, cholesterol cannot form bilayers on its own. Use the structure of the molecule to explain why. 4. Explain how the structure of phospholipids is the basis of the major properties of the bilayers that they form: physical form of the bilayer, self-sealing property, selective permeability, and fluidity of the bilayer. 5. Why are lateral movements of phospholipids in a bilayer so much easier than “flips” from one leaflet to the other? Explain why this means that we sometimes call a biological membrane a “two - dimensional fluid.” 6. Describe how fluorescence recovery after photobleaching (FRAP) works and list the types of scientific questions that can be answered using this technique.

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© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 7 of 11 Topic 2.2 - Practice Problems (Data analysis & problem solving. More likely to be exam-style) Problem 2.2.1 (Walkthrough Available) Lipid nanoparticles are a key component of the mRNA vaccines that were developed to help promote herd immunity during the current COVID-19 pandemic. They are based on a simpler system, known as a liposome, that was first developed in the 1980s. Liposomes are artificial vesicles, made of phospholipids. They can be prepared in the lab, with specific drugs on the inside. This allows a drug that would not otherwise be able to penetrate its target cell to deliver these compounds through fusion with the plasma membrane. A researcher you work with has discovered two novel compounds that have anti-tumour activity. She would like you to insert the drugs (known as Compound A and B) into 2 separate liposomes, in order to facilitate delivery to human cells. Assume that you have the expertise required to encapsulate the drugs (get them into the liposome). Based on the structure of each compound, draw where in the liposome each drug would be localized in the final product. For simplicity, use a triangle or square to represent each drug. Along with your drawing, provide a brief explanation of why you chose to locate the molecule where you did in the liposome. Problem 2.2.2 Martiniere et. al. (2012) tagged two membrane proteins (AGP4 and GPA1) with GFP so that they could be detected in cell membranes. After tagging, they carried out FRAP experiments to examine the mobility of AGP4 and GPA1. The photobleached region of the cell membrane is indicated by an arrow. Describe what you observe in each panel (A – D). a) When you compare all panels together, what can you conclude about AGP4 and GPA1 mobility? b) What could account for the differences in mobility between AGP4 and GPA1 that you observed? c) Draw the FRAP curves that you would expect to see for these two membrane proteins

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 8 of 11 Problem 2.2.3 Calcium ions (Ca 2+ ) are often used by the cell to modify the behaviour of other molecules. Here, Zilly et al. (2011) conducted an experiment to look at the effect of Ca 2+ on the mobility of the transmembrane protein Syntaxin 1. Fluorescently labelled Syntaxin 1 was expressed in cells and FRAP experiments were carried out in the presence or absence of Ca 2+ . a) Describe the change in fluorescence recovery of cells with and without calcium ions. b) Which treatment is experimental control? Explain. c) Based on the results, what can you conclude about the effect of Ca 2+ on the mobility of Syntaxin 1? Problem 2.2.4 RasGTPase (Ras) is a lipid-linked, plasma membrane-associated protein involved in cell signalling. Shawn Goodwin and colleagues did a study where they tagged Ras with GFP and expressed it in cells to examine its mobility using Fluorescence Recovery After Photobleaching (FRAP). They compared the mobility of Ras in the presence (+) or absence (-) of a drug called methyl- β - cyclodextrin (MβCD) that removes cholesterol from the membrane. Data adapted from Goodwin et.al. 2005. Biophysical Journal . Vol. 89:2 1398-1410 a) Compare the FRAP recovery in the upper and lower sets of images. Make sure to indicate which one is the control and how fluorescence recovery is influenced by the test condition. b) Based on the data in the images, draw what you would expect the fluorescence recovery curves for each condition to look like? Draw and label both curves on the axis below, as well as labelling the x and y axis. c) Based on this data, explain what you can conclude about how the mobility of Ras within the plasma membrane may be regulated. d) Predict how the mobility of Ras might differ in an artificial lipid bilayer (made entirely of phospholipids) compared to a cellular plasma membrane? Explain.

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 9 of 11 Practice Problems Walkthroughs Topic 2.1 – Chemical Features of Biological Membranes Problem 2.1.1 Example Answer: Orange Text , Instructor Comments: Blue Text The experiment described here is extremely important in the history of cell biology as it provided the first decisive evidence that the folding of proteins was dependent only on the primary structure of the protein and that it was a result of molecular self-assembly (i.e. spontaneous folding based on chemical properties). A Nobel Prize was awarded for this work in 1972. Background: This experiment deals with the structure and activity of ribonuclease, an enzyme that degrades RNA. It is produced in the pancreas. It is one of the first proteins for which the amino acid sequence was known. The protein's enzyme activity occurs only when the molecule is properly folded. As the molecule is denatured, the enzymatic activity is lost. The molecule has 4 disulfide bonds (C- S-S -H) that hold the chain together (see picture on the right). These form between cysteine residues. If the molecule is to be completely denatured, these bonds must be broken. Disulfide bridges are broken by placing the protein in a solution of 8 M urea and beta mercaptoethanol (HS-CH 2 -CH 2 -OH). The urea interferes with formation of hydrogen bonds and the mercaptoethanol (= reducing agent) provides a reducing environment, in which the disulfide bond will break, leaving 2 – SH groups (one on each cysteine). The plot on the right (Figure 1) shows how the enzymatic activity decreases as the mean number of disulfide bridges per ribonuclease molecule decreases during denaturation. Each disulfide bridge produces two SH groups: -C-S-S-C- → C-S H H S-C- (oxidized) (reduced) f) Describe how enzyme activity changes as the mean number of disulfide bridges decreases. Why do you think the enzyme activity is affected this way? Describe the Results: In the first part of this question, you are being asked to demonstrate your correct understanding of the data by describing, in your own words, the results shown in Figure 1. You are simply providing a written representation of the data.

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© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 10 of 11 Provide a Cell Biology Interpretation: The second part of this question is asking you to make some connections between the results in the figure and the bigger cell biology impact. What does this data tell us about enzyme activity? What can we infer from the data? Example Answer: As the number of disulfide bridges decreases (are broken apart), the % enzyme activity decreases. This indicates that protein folding is important for enzyme activity. As the S-S bridges are removed, the molecule unfolds (denatures) and enzyme activity is lost. If the reducing agent and the urea are removed the protein will slowly oxidize in the presence of air. The S-S bonds will reform and enzyme activity is restored, as shown in the Figure 2 on the right. g) Compare the changes in enzyme activity and reformation of S-S groups change as reoxidization progresses. What does this tell you? Describe the Results: In the first part of this question, you are being asked to demonstrate your correct understanding of the data by describing, in your own words, the results shown in Figure 2. A good answer will describe both the change in S-S groups and enzyme activity, and how they differ. Be sure to give a thorough description. Provide a Cell Biology Interpretation: The second part of this question is asking you to make some connections between the results in the figure and the bigger cell biology impact. What does this data tell us about enzyme activity? What can we infer from the data? Example Answer: As the S-S bonds reform, the enzyme activity begins to recover, fully recovering by 1200 minutes. We can also see that restoration of enzyme activity lags behind S-S bond reformation. This tells us that as the S-S groups reform, the enzyme begins to function again. h) What are some of the assumptions we are making in this experiment? There are a few assumptions being made. Example Answer: We are assuming that the whole protein is refolding properly, but maybe only the active site is still working i) Why do you think there is a lag between the formation of S-S bonds and the restoration of enzyme activity? Example Answer: I think the lag occurs because it takes time for the protein to refold before the enzyme can work properly j) Based on the data presented here, what did this landmark research contribute to our understanding of protein folding? Example Answer: Based on this data, it suggests that the primary amino acid sequence holds all the information needed to fold properly and spontaneously. It also suggests that proteins can be denatured and refolded. (Source of data: Anfinsen et al. 1961 PNAS 47, 1309, and White 1960 J. Biol. Chemistry 235, 383)

© 2024 – BIOL200 UBC Last updated 2024-01-04 12:11:00 AM Page 11 of 11 Topic 2.2 – Maintaining Fluidity in the Membrane Problem 2.2.1 Example Answer: Orange Text , Instructor Comments: Blue Text Lipid nanoparticles are a key component of the mRNA vaccines that were developed to help promote herd immunity during the current COVID-19 pandemic. They are based on a simpler system, known as a liposome, that was first developed in the 1980s. Liposomes are artificial vesicles, made of phospholipids. They can be prepared in the lab, with specific drugs on the inside. This allows a drug that would not otherwise be able to penetrate its target cell to deliver these compounds through fusion with the plasma membrane. A researcher you work with has discovered two novel compounds that have anti-tumour activity. She would like you to insert the drugs (known as Compound A and B) into 2 separate liposomes, in order to facilitate delivery to human cells. Assume that you have the expertise required to encapsulate the drugs (get them into the liposome). Based on the structure of each compound, draw where in the liposome each drug would be localized in the final product. For simplicity, use a triangle or square to represent each drug. Along with your drawing, provide a brief explanation of why you chose to locate the molecule where you did in the liposome. Instructor note: Your answer should include a drawing, since the question asks you to draw. There would be marks associated with that if its explicitly stated. However, my Word drawing abilities are limited, so I will only complete the written statement here (If someone creates a good drawing and posts it, I can include it in next year’s walkthrough!). Compound A is made of only carbon and hydrogen atoms. This means that I would expect it to be a non- polar molecule. As such it would ‘prefer’ to interact with the hydrophobic tails of the liposome phospholipids, in the interior of the bilayer. Compound B has both oxygen and nitrogen atoms, so it is polar. However, there are not areas that look like they could become charged. The molecule is small, so it probably would pass through the liposome membrane, and end up in the interior of the liposome.

Unit2A Problem Set (1)

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