Biochemistry

Biochemistry This assignment explores the relation between protein structure and function using cysteine proteases as example enzymes. In this assignment, you will use various online databanks and information gleaned in your textbook as well as other sources to learn how the three-dimensional conformation of enzymes contributes to their function, and you will then answer questions relating to what you have read or seen. This assignment introduces such concepts as “protein structure”, “protein topology”, “protein architecture” and an “enzymatic active site”. It also will expose you to important databanks involved in structural biology such as the Protein Data Bank (PDB: http://www.pdb.org, RCSB PDB: http://www.rcsb.org/pdb/home/home.do), the first stop for anything relating to protein structure. By familiarizing you with key concepts related to protein structure, this assignment serves to jump-start your learning of material which future lectures and reading will present in greater detail. We will also return to the use of databanks in biochemistry and molecular biology later in the semester. You may consult any reference or paper you wish and work with your fellow students. However, please cite any reference you consult, acknowledge anyone with whom you discuss this assignment and make sure your submitted work is in your own words. If you did not work with anyone on this assignment, include a statement to that effect in lieu of acknowledgements. You may write your answers in between the questions and use/add whatever space you need. By the way, the sequence tab in the PDB changed a few years ago. They made the output a lot snazzier looking, but it is a bit harder than it used to be to identify the sequence of the longest helix, which you will need to do in one of the problems in the attached assignment. What you will need to do is visually identify the longest bar in the row labeled “helix”, left click it (to select it) and then use your mouse scrolling function (or equivalent scrolling function, such as using two fingers on a touch pad) to zoom in (it seems to be that scrolling down zooms in): if you zoom in enough, you will see a sequence, with the helix you selected being shaded. The sequence does not seem to be selectable, so you will need to copy it down by hand and then type the sequence into a helical wheel generator. For what it’s worth, it is very easy to tell if your protein has a disulfide bridge (cystine), which is another task the assignment asks you to do: it is now a row in the sequence tab. If your sequence tab does not have a row labeled “disulfide bridge”, your protein does not have any cystine residues. 1. Your boss is studying a cysteine protease present in lysosomes and asks you to make 1.00 L of a 0.100 M acetate buffer at pH 6.00. You have powdered sodium acetate and liquid acetic acid (also distilled water and all the usual lab equipment) available. When finalizing your answer, think about how best to measure the various components of the buffer. Note that the pKa of acetic acid is 4.76. a. What do you mix in what quantity to make the buffer? In answering this question, think about the most practical way to measure each of the materials you need to make the indicated buffer, and convert your answer to units that reflect how you would dispense your buffer components. (5 pts) b. In testing various buffers, you added 0.010 millimoles of a strong acid to a 1.0 ml sample of your 0.100 M acetate buffer at pH 6.00 (hint: do not calculate the pH obtained after adding 0.010 millimoles of strong acid to a 1.0 ml sample of 0.200 M acetate buffer at pH 4.76), what is the new pH (of the buffer + the acid)? (3 pts) c. For comparison, what is the pH of a 0.010 M aqueous solution of acetic acid? Of 0.010 M perchloric acid (a strong acid)? (3 pts) d. There are specific reasons for using an acetate buffer to study the protease you are using; however, it may not be an optimal buffer for use at pH 6.00 in general. Which would be the best buffer to use at pH 6.00 in most cases, acetate buffer, formate buffer (pKa of formic acid is 3.75) or MES buffer (pKa of MES is 6.15)? (1 pt) 2. Plot for H2O, H2S, H2Se and H2Te a particular (measurable) physical or chemical property (e.g. density of the liquid, boiling point or pKa) as a function of molecular mass. Is there a trend (e.g. does the value of the property increase or decrease with molecular mass)? Does the value of that property for H2O fall on or near the trend line (or curve) or is water anomalous? Does water behave as if it were lighter than it really is or if it is heavier? Explain your answer to the last question in terms of hydrogen bonding. (5 pts) 3. Many viruses produce long “polyproteins” that are subsequently, hydrolytically cleaved into multiple, functional or structural proteins. The viral proteases catalyzing this cleavage are thus attractive targets for antiviral drugs. Many viral proteases, including the papain-like protease of SARS-CoV2 (c.f. the PDB “Molecule of the Month” article at https://pdb101.rcsb.org/motm/242) fall into a classification of proteins called “cysteine proteases”. For many of the remaining questions in this assignment, chose a cysteine protease whose structure is in the PDB. Answer the following questions based on your reading of a review on cysteine proteases that I will post on Blackboard as well as based the information you find on the PDB page (note the PDB page for your protein will have multiple tabs, such as a “Sequence” tab, which you should explore) for your chosen protein: a. What is the PDB ID of the protein you chose? (1 pt) b. How many residues does it have? What is its molecular mass? (2 pts) c. Are there any disulfide bonds (“cystine” residues upon hydrolysis) in the protein you chose? What does your answer say about where you would find the protein you chose: inside or outside a cell? (3 pts) d. What is the biological/physiological function (i.e. what does it do biologically or physiologically for the organism that makes it) of the protein you chose? What particular kind of chemical reaction does it catalyze? (2 pts) e. What other biological/physiological functions do cysteine proteases other than the protein you chose, have? (4 pts) 4. What is the pI (isoelectric point) of the protein you chose in the previous problem? What is the pI of the dipeptide cleaved from Angiotensin I to produce Angiotensin II: sequence HL? You may use ExPASy’s pI/MW calculation server (http://web.expasy.org/compute_pi/) to obtain the pI for the protein you chose. However, for the dipeptide HL, please calculate the pI by hand and show your work: you may use the pKa values on Wikipedia https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#General_chemical_properties https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#Side-chain_properties (you may need to copy and paste this link into your browser as ctrl-clicking it may not work) for your calculations – as always with this sort of thing, please cite which tools/tabulations you use. (5 pts) 5. Go to the Enzyme Nomenclature database on SwissProt (https://enzyme.expasy.org/) and search “by description (official name) or alternative name(s)” of the molecule you chose in problem #3. Click on the EC # (of the form x.y.z.w) for the protein you chose to learn about the reaction catalyzed by your chosen protein in order to answer the following questions: a. What is the enzyme class (EC #) of the molecule you selected? (1 pt) b. What is its preferred substrate? (1 pt) 6. Read about the concept of “protein topology” (c.f. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2144300/pdf/10211836.pdf for a description). What is the CATH (http://www.cathdb.info/ … note that clicking on this link might not work: you may need to copy the link to your browser) classification for each domain (even if there is more than one domain, each may have the same classification) in your protein? Note that the CATH classification for each domain (which will be given a domain ID based on a PDB ID) will be formatted as 1.20.30.40 (and indicated as a “Superfamily”)? What does “CATH” stand for and what is meant by the term “architecture” and “topology”? You may also wish to also look at the Annotations tab on your protein’s PDB page and compare the classifications. E.g., does each classification method parse your protein into the same number of domains? (4 pts) 7. Look at the sequence tab for your protein’s PDB entry. Copy the sequence (you may need to enter the sequence by hand) of the longest helix (not the entire protein sequence … just the longest helix; you may need to enter the sequence by hand into the helical wheel generator’s website) into a helical wheel generator such as the one found at https://heliquest.ipmc.cnrs.fr/cgibin/ComputParams.py (at that site, you will select -helix for the helix type and “FULL” for the window size). Copy/paste the helical wheel into your assignment (or include a print-out of the helical wheel if you are handing in a hard copy of the assignment). Additionally, annotate your helical wheel: (A) indicate the hydrophobic side and hydrophilic side of your helix and (B) indicate any “helical capping” residues, including (i) any P,N,Q,D or E among the first four amino acid residues in your helix (ii) any positively charged residues 3 residues away from a negatively charged residue (iiI) any positively charged residues near the C-terminus of your helix. Are there any prolines in your helix outside of the first four (N-terminal) residues? (5 pts) 8. Study in your textbook Fig. 6-22, 6th edition / 6-27, 8th edition and read the related discussion in the body of your text (which also provides a good introduction to what I mean by “mechanism”) about the mechanism by which serine proteases catalyze hydrolysis. a. Briefly describe one or more key differences between serine proteases and cysteine proteases (2 pts) b. Note (from your answer to problem 3b) that cysteine proteases are giant (macro certainly applies here) molecules. In some cysteine proteases, the catalytically important cysteine residue giving this class of proteases its name is separated from other active site residues by over one hundred amino acids in the protein’s sequence. Suppose a biochemist produced a short peptide, which contained all of the active site residues of a cysteine protease but which lacked protease activity. Which of the following are reasonable hypotheses (note: even if only one of these hypotheses happens to be true in explaining a particular short peptide’s lack of activity, indicate all reasonable hypotheses in answering this question) as to why that short peptide lacked activity? (3 pts) i. The short peptide lacked well-defined binding pockets to keep (only specific) substrates in place and facilitate conformational distortions toward the configuration of the transition state ii. The short peptide had multiple active sites but the full-length protein had even more active sites on each polypeptide chain iii. The short peptide lacked a hinge region all-together, so it could not “cut” its substrate iv. The short peptide had insufficient flexibility to facilitate conformational distortions toward the configuration of the transition state v. The short peptide had only one active site while the full-length protein had multiple active sites on each polypeptide chain vi. The side chains of adjacent residues on the short peptide pointed toward each other (and hence formed a functional active site) only in conformations of the peptide that lead to gauche or eclipsing interactions between those side chains

Biochemistry

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