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)

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.

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