Homework before the lab

How many RNA-binding domains are enough?

Evolution of dsRBD architecture in ADAR proteins. Before you come to the lab, get to know the plasmid we will express the proteins from, and learn to predict protein properties from sequence. Everything is computed right here, in your browser.

Anna Perederina, Arina Mazurova, Artur Safin, Max Marchenko

Tasks

Answers are checked on this page; numbers are compared with a tolerance. Nothing is sent anywhere — this is a trainer, not an exam. Your answers are kept in this browser.

A. Getting to know the vector

1. Open the plasmid map. What is the size of the vector in base pairs?
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2. Which antibiotic should you use to select bacteria carrying this plasmid?
   — choose — ampicillinkanamycinchloramphenicoltetracycline Check
   Hint

   Find the resistance gene on the map and read its description in the feature list.
3. How many base pairs does the MBP coding sequence span, and how many amino acids is that?
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   Hint

   Click MBP on the map — the coordinates fill in and the fragment length appears under the sequence.

B. Computing protein properties

4. Open the calculator and load the ready-made MBP sequence. What are its isoelectric point and molecular weight in kDa (round to one decimal)?
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   Check yourself

   Paste the same sequence into ExPASy ProtParam — the numbers must match.
5. Now pick “Human, ADAR2 — dsRBD1” from the ready-made sequences and compute the pI of a single domain.
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6. Compare the pI of a single dsRBD with the pI of MBP. What charge will each of them carry in a buffer at pH 7.5?
   — choose — both positive dsRBD positive, MBP negative dsRBD negative, MBP positive both negative Check
   Why

   The RNA backbone is negatively charged, and the dsRBD surface that contacts it is rich in lysines and arginines — hence the high pI. MBP binds no nucleic acid and its pI is around 5, so at pH 7.5 it is negative. The same difference is what ion-exchange purification exploits.

C. Choosing a buffer

A buffer works within pKa ± 1, and the working pH is taken 1–1.5 units away from the protein's pI. More detail is in the Reference tab.

7. You made Tris-HCl pH 8.0 at room temperature (25 °C) and moved the column into the cold room (4 °C). What will the pH of this buffer become? For Tris, ΔpKa/ΔT = −0.028 units per degree.
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8. The binding buffer needs 5 mM MgCl₂. Which buffer will not do?
   — choose — HEPESTrissodium phosphateMOPS Check
   Hint

   One of these anions forms an insoluble salt with divalent cations.
9. Which buffer from the table would you take for a working pH of 4.8?
   — choose — sodium acetateMESPIPESTris Check
10. The N-terminal construct of human ADAR2 has pI 7.11. You want to bind it to an anion exchanger, so the protein must be negatively charged and the pH must sit one to one and a half units above the pI. Which buffer fits?
    — choose — sodium acetate, pH 4.8MES, pH 6.0 sodium phosphate, pH 7.2Tris, pH 8.5 Check
    How to reason

    First pick the pH: it must be 1–1.5 units above the pI, so roughly 8.1–8.6. Then find a buffer whose pKa ± 1 window covers that pH. Phosphate at pH 8.2 is already at the very edge of its range and buffers poorly.

D. Evolution of the architecture

11. The table at the bottom of the “Protein” page lists the N-terminal constructs of all our organisms. Which of them has the most dsRBDs?
    — choose — Human, ADAR1 p150Human, ADAR2 Fruit fly, AdarSquid, ADAR2 Hydra, ADAR Check
12. How many dsRBDs does human ADAR1 have?
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13. And which protein in our set has just one dsRBD?
    — choose — Human, ADAR2Fruit fly, Adar Squid, ADAR1Hydra, ADAR Check
    Note

    This protein is the longest of our N-terminal constructs, yet almost all of that length is unstructured, and it carries a single RNA-binding domain.