F21_MCDB108A_FinalExam_key (2)

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MCDB 108A – Final Exam – Dec. 8, 2021 NAME:_______________________ PERM:______________ By writing your name you attest that you neither have nor will receive or give unauthorized aid on this assessment. Use the information here to answer the following 6 questions. The MAPT gene encodes for tau protein in humans. Alternative splicing of MAPT produces 6 naturally expressed tau isoforms. The longest isoform of human tau protein expressed in cells contains all four microtubule binding repeats (4R) and a long (L) projection domain due to the inclusion of both N-terminal inserts. This “4RL” tau isoform is composed of 441 amino acids. There are 106 non-polar, 117 uncharged polar, 56 acidic, 70 basic, 49 Gly, and 43 Pro residues (note there is no group overlap here). 1. (4pts) You wish to recombinantly express 4RL in bacteria to produce and isolate large quantities of it for some experiments. Which of the following components must be included in your expression vector in order to express and purify tau? Select all that apply. (– 0.5pts for each incorrect answer selected) a. Origin of replication b. Anti-biotic resistance gene c. Bacteriophage resistance gene d. Entire MAPT gene e. MAPT gene excluding introns f. MAPT gene excluding exons g. MAPT promoter sequence h. MAPT termination sequence i. Bacterial promoter sequence j. Bacterial termination sequence k. GFP tag for protein localization l. Poly-His tag for isolation 2. (2pts) Once expressed, you lyse and boil your bacterial cells to create a homogenized cell lysate for centrifugation. After a few rounds of centrifugation, you collect a fraction that contains your a mixture of tau and other cytosolic proteins. You want to use column chromatography to separate 4RL from the other proteins. Which chromatography and elution buffer selection would work best as a first step? a. Cation-exchange chromatography with a running buffer of increasing NaCl concentration over time b. Cation-exchange chromatography with a running buffer of decreasing NaCl concentration over time c. Anion-exchange chromatography with a running buffer of increasing NaCl concentration over time d. Anion-exchange chromatography with a running buffer of decreasing NaCl concentration over time

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 2 3. (4pts) After further purification steps, you are confident that you have a solution containing only your tau, because SDS-PAGE shows only one band near the expected mass of 4RL (expected mass is ~ 50 kDa). You excitedly show your lab mate your successful result, but your lab mate is quick to point out that your protein band does not align with the 50 kDa marker in your protein MW ladder. As it turns out, other highly charged, intrinsically disordered (no globular structure), highly soluble proteins like this tend to also run differently in SDS-PAGE. Assuming you have expressed, purified, and isolated the protein exactly as expected, and you set up and ran your SDS-PAGE correctly, why might tau or other similar proteins not run at the expected MW in SDS- PAGE? Your answer should be concise (no more than 4 sentences) and must address both the protein properties and the mechanisms of SDS-PAGE for full credit. Few hydrophobics / lots of charged residues, so SDS doesn’t interact with it like most (globular) proteins and the native charges on the protein all make the electrophoretic forces slightly abnormal compared to other proteins. 4. (2pts) You have convinced yourself that the SDS-PAGE result in problem 3 is not an issue, but your research advisor is adamant that you verify that the protein is unaltered and the sequence is correct. What technique would you use to verify that your tau has been correctly expressed and purified without having been modified in the process? Tandem mass spectrometry would answer both of those questions in one go

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 3 5. (4pts) Tau-tubulin interactions are thought to be facilitated in part by electrostatic attractions between tau and the MT surface. A certain post-translational modification to a residue on tubulin’s C-terminal sequence (which is exposed on the microtubule surface) is common, and you’re curious if it affects tau-tubulin binding. Similarly, a modification to a residue on tau near the MTBD is very common, and you wish to see if that change to tau, independent of any modifications to tubulin, can affect tau-tubulin binding. You perform protein binding assays with the following three systems: (1) unmodified tubulin and unmodified tau; (2) modified tubulin and unmodified tau; and (3) unmodified tubulin and modified tau. Data for these three experiments are shown below. Which of the following conclusions can be made based on the information provided? Select all that apply. (– 0.5pts for each incorrect answer selected) a. The apparent tubulin-tau dissociation constant (K d ) in each of the modified systems (2 and 3) is approximately half that of the unmodified system (1) b. The apparent tubulin-tau K d in each of the modified systems (2 and 3) is approximately double that of the unmodified system (1) c. Both modifications appear to reduce binding affinity between tau and tubulin d. Both modifications appear to increase binding affinity between tau and tubulin e. The type of modification on tubulin in (2) is likely the same as the type of modification of tau in (3) f. The type of modification on tubulin in (2) is likely different from the type of modification on tau in (3)

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MCDB 108A – Final Exam – Dec. 8, 2021 of 13 4 6. (2pts) Which of the following pairs of post-translational modifications would most likely account for the observed phenomenon in problem 5? Modified amino acids are shown below. Note that the pKa values of the two phosphate hydroxyls are approximately 3 and 7. Assume your experiments take place at pH 7. a. Methylated Glu on tubulin, phosphorylated Ser on tau b. Phosphorylated Ser on tubulin, methylated Glu on tau c. Acetylated Lys on tubulin, methylated Glu on tau d. Methylated Glu on tubulin, methylated Glu on tau e. Phosphorylated Ser on tubulin, phosphorylated Ser on tau

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 5 7. (4pts) Methyl Accepting Chemotaxis Proteins (MCPs) are bacterial inner membrane proteins that detect external stimuli and modify swimming behavior to promote bacterial movement towards attractants and away from potential toxins in their environment. MCPs are typically purified as 200 Å long α-helical coiled-coils. In addition to sensing external stimuli, a cluster of Glu residues in the cytoplasmic portion can be methylated/demethylated to alter the signaling behavior of the receptor. Each Glu in this cluster is approximately 4 residues apart in the peptide sequence. Provide a reasonable explanation why methylating the carboxyl group of the Glu may alter the function of the protein. (Hint: These Glu residues do not directly interact with any other proteins involved in signaling) Methylation will remove the charge from the Glu residues. Clusters of negative charges on an alpha helix can destabilize it significantly. Methylation is likely shifting the molecule from an unstable alpha helix (or disordered protein) to a stable alpha helix which will alter the function of the protein 8. (3pts) In order to study MCPs in the lab, we often want to purify them in a homogenous state (i.e. all MCP are altered - or unaltered - to the same extent), but the bacteria can alter them in ways we are not able to control. Instead of isolating native (or wild-type) MCPs, we need to make genetic substitutions to mimic different states of the protein that the bacteria will not modify. Which of the 20 canonical amino acids would best substitute for Glu that would mimic the methylated Glu found in the MCPs. Briefly explain your reasoning (Methylated-Glu shown in for reference). The methylated Glu still has its carbonyl group, giving it some level of polarity. Glutamine would be the most similar canonical amino acid as it has the carbonyl group, some polarity, but no charge.

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 6 9. (3pts) Triacylglycerols (TAG) are stored in unique cellular compartments due to their unique biochemical properties. These organelles are essentially huge, membrane-bound collections of TAG. What unique feature do you think the membrane of these organelles would have compared to other organelles? Why? Unlike most other cellular compartments/extracellular space, the interior of lipid droplets would be highly non-polar. Thus, the interior of the membrane would need to be hydrophobic. Instead of using a bilayer, lipid droplets would be better served by a monolayer that leaves the hydrophobic lipid tails to interact with the hydrophobic TAG. 10. (3pts) Most proteins that associate with the membrane of the TAG organelle are transmembrane proteins. A few of these proteins have extensions into the inner space of the organelle. What would you expect to be different about these TAG-membrane proteins compared to a typical protein in the cell? These proteins would expose more of their hydrophobics to the interior of the membrane as well as the interior of the lipid droplet with very little hydrophilic residues due to the hydrophobic nature of the TAG interior. 11. (4pts) Below are 4 proteins you have in a mixture that you are attempting to separate using column chromatography. Describe the three sequential steps you might take to separate them from each other. For each step, describe what you expect to elute together, what you expect to elute separately, and which fraction each protein should be found in. (e.g. “using x chromatography, proteins a, b, and c will elute together in an early fraction, while protein d will elute separately in a later fraction”) Protein: Automata Halo Hades Cookie-Run Molecular Weight (kDa) 13.22 14.14 23.87 13.87 Net charge at pH 7 – 8.32 + 0.97 + 1.26 + 1.48 Glucose binding Tight binding No interaction No interaction Tight binding Anion exchange chrom: Halo, Hades, and Cookie-run elute earlier while Automata stays on the column then comes out after elution Size Exclusion chrom of results: Hades elutes early while Halo and Cookie-run come out later, together Affinity chrom with Glu column of results: Cookie-run binds to column while Halo elutes early. Then wash out Cookie-run from column

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MCDB 108A – Final Exam – Dec. 8, 2021 of 13 7 12. (2pts) Which single characteristic is common to both DNA and RNA polymerases? a. The nucleotide substrates they incorporate b. Their requirement for a primer c. Their error rate d. The type of chemical reaction they catalyze e. Their processivity 13. (3pts) DNA synthesis by DNA Polymerase III occurs on both the lagging and leading strands created at the replication fork. At the front of this replication fork is DnaB helicase, a protein that forms a ring-like structure with a central hole through which DNA is threaded. As DNA passes through DnaB helicase, the parent duplex is unwound. The b sliding clamp protein forms a similar ring-like structure, binding the primed lagging strand within its central hole and enhancing the processivity of the polymerase III complex by preventing its dissociation from the DNA. Of these two “donut-like” proteins that bind DNA, which is likely to have a more narrow central hole? Helicase or sliding clamp? Choose one and provide a one-sentence justification. Helicase would have a more narrow hole because it needs only to bind a single (lagging) strand of the parent DNA as it moves forward to unwind the duplex. Sliding clamp binds the duplex of primer and lagging strand, therefore requiring space for double-stranded and not single-stranded DNA. 14. (4pts) RNA polymerases can transcribe genes from single strands of DNA, with either strand able to serve as a template for transcription. Which of the following statements about RNA polymerase are TRUE? Select all that apply. (– 1.0pts for each incorrect answer selected) a. Transcription occurs in the 5’ to 3’ or the 3’ to 5’ direction, depending on which strand the gene is encoded b. Proteins bound to the promoter region of a gene tend to have little effect on transcription c. Proteins bound nearby to the promoter region of a gene tend to have little effect on transcription d. The sequence of nucleotides on the coding strand is the same as that of the RNA being transcribed (aside from differences between RNA and DNA)

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 8 15. (2pts) An individual is suspected to have Andersen’s disease, a condition characterized by a deficiency in the glycogen branching enzyme. Which of the following downstream effects would this enzyme deficiency cause? Select all that apply. (– 0.5pts for each incorrect answer selected) a. Glycogen devoid of a -1-6 linkages b. Quicker breakdown of glycogen in the cardiac muscle c. Decreased glycogen solubility d. Decreased storage of glucose e. High blood sugar 16. (3pts) Shown below is a graph of the enzymatic activity of wild type glycogen branching enzyme and a deficient glycogen branching enzyme. Based on your knowledge of Michaelis-Menten kinetics, which curve represents the deficient enzyme? Why? Curve B would represent the deficient enzyme. Km=[S] at 1/2Vmax Curve A has a higher Vmax, so it would have a lower Km, and would have a higher affinity. Curve B would have a higher Km, which means it would have a lower affinity for it’s substrate. 17. (2pts) Consider an in vitro experiment in which all components needed for protein synthesis are present. If radioactively labeled amino acids are added (in addition to non- radioactive labeled amino acids already present), in which of the following components could you find radioactivity if you were to “magically” halt all activity and take a microscopic look at the components in your system? Select all that apply. (– 0.5pts for each incorrect answer selected) a. mRNA present before adding the labeled a.a. b. mRNA formed after adding the labeled a.a. c. aminoacyl tRNA synthetases d. actively translating ribosomes e. peptides

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 9 18. (2pts) Use the segment of mRNA sequence below. Taking into consideration the Wobble hypothesis and the possibility for frame shifts, how many unique amino acids could be synthesized? (e.g. if multiple Gly residues can be synthesized, that’s only one unique amino acid - Gly) GUGUGUGUGUGUG a. 0 b. 2 c. 4 d. 9 e. 13 19. (3pts) We have discussed at length the role of alpha helices and certain beta-sheet structures that enable proteins to embed themselves in membrane bilayers either completely or partially. Proteins that are partially embedded must pass from the non- polar environment of the bilayer to the polar environment outside. The length of the peptide bond and the size of this polar–non-polar “interface” effectively requires an amino acid residue to reside at this interface. Which of the following amino acids would you expect most stably bridges the polar–non-polar interface? Leu, Trp, Cys, Thr, or Lys? Justify your answer. Lys could be justified by the length of its non-polar side chain that terminates with a polar, charged group. The non-polar region could reside in the more non-polar side of the interface, extending the polar end of the side chain out to the polar side of the interface. Trp could be justified by the size of its aromatic ring structure, which can reside in the non-polar environment and the hydrogen bonding potential of N of the side chain indole would allow it to interact favorably with water.

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MCDB 108A – Final Exam – Dec. 8, 2021 of 13 10 20. (3pts) You have isolated a protein you think is involved in DNA polymerization, and you want to test the hypothesis that it is in fact a DNA polymerase. To test this, you make reaction mixtures containing your isolated protein, free nucleotides to be used in the synthesis reaction, a template DNA strand that is 50 nucleotides long, and a primer that is 13 nucleotides long. To test whether DNA synthesis has occurred during your reaction, you plan to run the components through gel electrophoresis to separate and visualize short and long strands. However, the template DNA would run at the same MW as any newly synthesized strand. To differentiate between template strands that are 50 nucleotides long and newly synthesized strands that are 50 nucleotides long, you decide to label your primer at a free hydroxyl. That way, any label detected at the 50 nucleotide MW band on your gel will indicate that DNA polymerization took place. On which end of your primer should you attach the label? Answer 5’ or 3’ and provide a one sentence justification. 5’ so that polymerization can still occur on the 3’ end. 21. (3pts) Suppose you ran your gel and saw no labeled DNA strands that were 50 nucleotides long and only saw labeled strands that were 13 nucleotides long (your primer). You tell your research advisor that the result conclusively shows your protein is not a DNA polymerase. Your advisor recommends that you run a control experiment to make sure your reaction mixtures would have allowed DNA polymerization had your protein been a polymerase. What change do you need to make to the components in the reaction mixture outlined above? Swap your protein with a known DNA polymerase and run the same reaction and the same gel.

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 11 22. (3pts) You treat cells with a chemical that induces mutations and isolate two mutant cell lines. One cell line produces protein X with a tryptophan in the position where a leucine normally occurred, and the other cell line produces protein X with a valine in that same leucine position. You treat each mutant cell line with the chemical one more time and find that both mutant cell lines now produce protein X with an glycine where the original leucine used to be. Assuming all changes were point mutations in the gene encoding protein X, what codon was used for leucine? For valine? For glycine? Use the codon table below. Circle one codon for each amino acid. Leu : UUA UUG CUU CUC CUA CUG Val : GUU GUC GUA GUG Gly : GGU GGC GGA GGG 23. (3pts) Referring to the problem above, do you think it would have been likely to go from Leu to Gly in a single mutagenesis step? Answer yes or no and explain your answer in one sentence. No.

MCDB 108A – Final Exam – Dec. 8, 2021 of 13 12 24. (3pts) The COVID-19 vaccines that were rapidly developed in response to the pandemic are lipid-based mRNA delivery vectors. The mRNA “payload” of these vectors encodes for a modified version of the viral “S” spike glycoprotein that is 1,273 amino acids long. While these mRNA vaccines mark the significant progress that biochemistry has made in understanding nucleic acid and immune response systems, the vaccines themselves have some striking drawbacks compared to “normal” vaccines. The most notable of these drawbacks is the storage requirements and shelf-life of the vaccines, with Pfizer’s version requiring storage at -70 C and with very low predicted shelf lives. Importantly, “naked” mRNA by itself in solution is chemically unstable, while the lipid component of these vaccines is known to be stable for significantly longer periods of time. On the other hand, lipid-based short interfering RNA (siRNA) vectors that deliver nucleic acid payloads less than 30 nucleotides in length have stable shelf lives (much more stable than the common COVID-19 mRNA vaccines). Consider the scenario where a lipid-based mRNA and a separate lipid-based siRNA vector are made using the same total mass of nucleic acid and the same mass and composition of lipids. Why might the siRNA system have a longer shelf-life (chemical stability) than the mRNA system? Shorter sequence length of siRNA system means lower probability of phosphodiester backbone auto-hydrolysis per siRNA molecule, increasing the probability of an intact siRNA to be delivered with the system. Similarly, with the same mass of nucleic acid in the two systems there is significantly more individual siRNA molecules, so even if hydrolysis of the backbone were to occur in some siRNA molecules, there would be plenty left as “back up”

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MCDB 108A – Final Exam – Dec. 8, 2021 of 13 13 25. (2pts) Which of the following fatty acids would you expect to have the highest melting point? a. b. c. d. 26. (6pts) Match each of the following structures to their corresponding name below. a. D-Fructose __ 5 __ b. D-Galactose __ 6 __ c. D-Glucose __ 1 __ d. a -D-Glucose __ 2 __ e. a -D-Mannose __ 3 __ f. b -D-Ribose __ 4 __

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