MCDB 310 Project 2 PDF

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Protein Sequencing/Analysis and RNA Genome Project #2 Spring 2020...

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MCDB 310, Spring 2020. Project 2

Krupa Patel - Section 107

General Instructions: 1. Students may work alone or in groups consisting of up to 4 students, all of whom are associated with the same GSI (not necessarily the same Discussion section). Groups should submit a single copy of their work with the names of all participants on it. You are responsible for making sure that the assigned person in your group includes your name on the project submission, and submits the project within the deadline. 2. Projects should be submitted to the Canvas assignments section of your MCDB 310 discussion section and NOT to the general site. This ensures that your project ends up with the correct GSI. The answers should NOT be submitted directly to Dr. Akey. (please see exception below for late submissions). The GSIs will grade them all and then post the scores in the Assignment folder. th 3. Deadline for submission is Friday, May 29 11:59 PM. Resubmissions, even if they are within the deadline, will not be accepted. We can consider submissions that are late but by no more than 24 hours past the deadline. However, these late submissions will be graded for no more than 1⁄2 credit (i.e. a maximum score of 13/25). These late submissions will have to be emailed directly to your GSI because Canvas will not accept late submissions. Submissions attempted after the 24-hour late period will NOT be accepted. 4. Expanding textboxes are provided for typed answers. For questions involving drawing, graph axes are available. Please make sure to answer all parts of each questions to receive full points. Please fully read each question before answering.

Question 1) Below is the structure of the COVID 19 main protease (Mpro). (Source: Zhang et al., 2020)

a) The structure above was determined from a crystal of COVID 19 Mpro. What technique (describe in lecture) was used to determine this structure? What is considered the most difficult step of this technique? (4 pt)

Answer: X-Ray Crystallography is the technique used to determine this structure because it requires crystallizing the proteins in order to collect diffraction data, calculate electron density, and fit the residues into the density. Proteins will solidify into crystals under certain conditions. These conditions are usually made up of salts, buffers, and precipitating agents. Thus, the first and most difficult step is to obtain an adequate crystal of the material under study. Hundreds of conditions varying the salts, pH, buffer, and precipitating agents are combined with the protein in order to crystallize the protein under the right conditions. b) Viral proteases are used to cleave protein precursors into their active forms. What type of enzyme regulation uses this mechanism (give the specific name used in lecture)? What is an example of an enzyme covered in class that uses this type of regulation? (4 pt)

Answer: Irreversible activation/modification uses this mechanism in the case of zymogens, which are inactive precursors of proteases that are cleaved into their active forms by viral proteases. An example covered in classes that uses this type of regulation is trypsinogen, which is activated by enteropeptidase, and thus, cleaved to its active form, trypsin. The pancreas stores the inactive form trypsinogen because the active trypsin would cause severe damage to the tissue of the pancreas.

c) COVID 19 is a cysteine (thiol) protease. Unlike the serine protease example in class, it has a catalytic dyad made up of Cys145 and His41 (shown above). Assuming its mechanism is otherwise similar to that of a serine protease, what are the most likely roles for the Cys145 and His41 residues in the cleaving of the peptide bond? (4 pt)

Answer: The likely role of the catalytic dyad (His-41/Cys-145) site is to attract the π electron density from the peptide bond Gln–Ser, increasing the positive charge on C(CO) of Gln and the negative charge on N(NH) of Ser, thereby, weakening the Gln–Ser peptide bond. The catalytic functional group is the imidazole group of His-41 and the S in Cys145. The N on the imidazole ring probably plays the acid–base catalytic role. In most serine proteases that have a catalytic Ser-His-Asp triad in their active sites, a stable water molecule would occupy the Asp position of a typical serine protease triad, and this molecule would play a role in stabilizing the imidazolium ring during catalysis. It’s suggestive that His41 acts as a general base during the diacylation step; more than likely, there’s a bridge water molecule near the catalytic dyad His-41 and Cys-145 and the peptide bond to be cleaved. The bridge water molecule joins the hydrolysis reaction and plays an important role during the process.

Question 2) Below is a multisubunit protein structure that contains the COVID 19 RNA-dependent RNA polymerase (made up of “Fingers”, “Thumb”, and “Palm”). (Source: Gao et al., 2020)

a) Unlike humans, which have DNA-dependent RNA polymerases, many viruses have RNAdependent RNA polymerases (to replicate their RNA-based genome without needing a DNA intermediate). Name two features of RNA that an RNA-dependent RNA polymerase could use to distinguish between an RNA template and a DNA template. (4 pt)

Answer: RNA and DNA have three structural features that distinguish themselves from each other: 1) RNA contains uracil while DNA contains thymine (uracil differs from thymine in that it lacks a methyl group on its ring). 2) RNA has the sugar ribose (makes it more reactive than DNA and unstable in alkaline conditions) while DNA has the sugar deoxyribose (contains one less hydroxyl group than RNA’s ribose). 3) RNA is single-stranded (with shorter strands and A-form helix) whereas DNA is double-stranded. RNA’s larger helical grooves mean it is more easily subject to attack by enzymes.

b) Give an example of an amino acid you might expect to find in the recognition site of an RNAdependent RNA polymerase and describe the corresponding feature of RNA it could interact with. (3 pt)

Answer: An aspartate residue of a motif in the recognition site of an RdRp would be involved in the selection of ribonucleoside triphosphates over dNTPs and, thus, help determine whether RNA rather than DNA is synthesized. This corresponds to the RNA feature of having the sugar ribose, unlike DNA, which has deoxyribose as the sugar. Mechanistically, the carboxylate side chain of aspartate could discriminate NTPs (in RNA) from dNTPs (in DNA) by forming an H bond with the hydroxyl group of the incoming NTP. c) Magnesium and/or manganese cations are common cofactors for RNA-dependent RNA polymerases. What feature of RNA could these cations balance/associate with? (2 pt)

Answer: Metal cofactors are critical for the nucleotidyltransferase reaction catalyzed by RdRps. RdRps catalyze RNA-template dependent formation of phosphodiester bonds between ribonucleotides, but this can ONLY happen in the presence of divalent metal ions. The divalent metal ions are crucial for polymerization reaction because they coordinate the catalytic aspartates and facilitate the formation of a phosphodiester bond between NTPs (feature present in only RNA). The metal cofactors help in positioning the NTP’s triphosphate group in an optimal position for attack by the sugar of the incoming strand (probably done by activating the 3 ′-OH for nucleophilic attack by lowering its pKa value and orienting the nucleotide substrate to stabilize the negatively charged transition state). d) Compared to a DNA-based genome, name four structural features you would expect to see in an RNA-based genome. (4 pt)

Answer: 1) RNA-based genome encodes the protein RNA-dependent RNA polymerase (RdRp), which is essential for the replication of both positive and negative strand ssRNAs. as well as dsRNAs. a. RNA-based genome is more susceptible to mutation during transcription than DNA-based genome during replication, because RNA polymerases lack the proofreading ability of DNA polymerases. 2) Typically, smaller than DNA-genome viruses. 3) Chemically somewhat unstable, most likely due to the extra OH (ribose sugar) in RNA and single strand structural feature. Genetic material also contains uracil as a base, not thymine (like in DNA). 4) Retroviruses are replicated as DNA following the conversion of the RNA into DNA by a reverse transcriptase. Also use enzyme called integrase, to insert the retroviral DNA into the genome of the host cell.

References: Gao, Yan, Liming Yan, Yucen Huang, Fengjiang Liu, Yao Zhao, Lin Cao, and Tao Wang et al. 2020. "Structure Of The RNA-Dependent RNA Polymerase From COVID-19 Virus". Science 368 (6492): 779-782. doi:10.1126/science.abb7498. Zhang, Linlin, Daizong Lin, Xinyuanyuan Sun, Ute Curth, Christian Drosten, Lucie Sauerhering, Stephan Becker, Katharina Rox, and Rolf Hilgenfeld. 2020. "Crystal Structure Of SARS-Cov-2 Main Protease Provides A Basis For Design Of Improved Α-Ketoamide Inhibitors". Science, eabb3405. doi:10.1126/science.abb3405....