C430 Exam 2 - Protein purification

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C430 Exam 2 - Protein purification
2013-03-07 23:21:04

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  1. 1. What if you discover a novel protein based on function, but don't know sequence?
    2. How are short polypeptides sequenced?
    3. What does primary sequence dictate? What is important about this?
    • 1. Protein seq can be derived indirectly from DNA sequences in rapidly growing genome databases.
    • 2. With automated procedures (Edman degradation) 99% accurate nowadays.
    • 3. Primary sequence dictates 2 -->3--> 4, so proteins with similar functions have similar structures and vice versa.
  2. 1. How are larger proteins sequenced?

    2. What are steps to determining a primary protein sequence?

    3. What can you do with info from homology?
    1. Must be sequenced in smaller segments, bc overall accuracy decreases as length increases. Must breakdown disulfide bonds with BME.

    2. Sequence genome, annotate it, and then determine protein sequence based on homology.

    • 3. Using info from homology, can guess putative function of protein.
    • 4.
  3. 1. What is the purpose of learning about Mb and Hb?
    2. Where is Mb found? What type of protein is it?
    3. What is Mb's function?
    4. What is its main secondary structure and how many does it have?
    • 1. To learn how protein tertiary and quaternary structure influence function
    • 2. Muscle - globular
    • 3. Mb's function is to store O2 until it can release it for ETC use.
    • 4. It has 8 a-helices
  4. 1. When is porphyrin ring system domed? When is it planar? (O2 conditions)
    2. Describe the coordination bonds to Fe2+. 3. How does heme bound to Mb differ?
    4. Where does iron lie in the ring system?
    • 1. Domed = no O2 (chrome to your dome = you're not going to live); Planar = O2 bound.
    • 2. Fe2+ can make 6 coordination bonds - 4 of which are bound to N in heme(planar), leaving two (perpendicular) free.
    • 3. In Mb, proximal His residue binds to one of the open coordination bonds, leaving the last one free to bind O2 (prevents two O2s from binding to Fe and causing it to turn into Fe3+
    • 4. Iron lies planar to ring system
  5. 1. Why do we need heme?
    2. Why do we need Mb and Hb? (2)
    3. Why do we use Fe to bind O2?
    1. We need heme to prevent Fe2+ from spontaneously oxidizing to Fe3+ --> Fe3+ is very dangerous --> free radicals.

    2. O2 is sparingly soluble gas - cannot travel long distances in blood without bubbling out of solution.

    So, we need iron (in the form of heme) to bind O2 and we need Mb and Hb to stabilize heme and prevent it from binding to CO, etc.

    2. No AA has suitable side chains to reversibly bind to O2, so Fe is best option.
  6. 1. What is ?
    2. What does it measure?
    3. What does a low Kd indicate?
    4. Why do we use PO2 instead of [O2]?
    5. Describe, in words, how Kd relates to saturation.
    6. Write equation for saturation for O2.
    • 1. Dissociation constant
    • 2. Can be used to measure affinity of protein for ligand (like Km)
    • 3. Low Kd = high affinity for ligand.
    • 4. Partial pressure is easier to measure for a gas than concentration.
    • 5. When [L] = Kd (PO2 = P50), 50% of protein is saturated.
    • 6.
  7. 1. Draw a picture of Mb's dissociation curve. How would you describe this curve?
    2. What does shape of Mb's dissociation curve tell us? (2)
    3. What does the plateau in the curve mean?
    • Rectangular hyperbolic

    2. (1) Only one binding site on protein and (2) Small P50 indicates that Mb has high binding affinity to O2.

    3. At plateau, it means that no matter how much more O2 is added, will not increase saturation bc there are no more free Mbs left.
  8. 1. Describe Hb's quaternary structure
    2. What is Hb's main function?
    3. Why can't Mb do this? (2)
    4. What does each subunit of Hb look like?
    • 1. Tetramer - four subunits (a1,2 and B1,2) each prosthetically bound to heme.
    • 2. To pick O2 up in O2 rich lungs and deliver it to O2-poor tissues.
    • 3. Mb is better as a STORAGE protein because (1) it's relatively insensitive to small changes in concentration of dissolved O2 and (2) has high affinity for O2 and will not be able to release as well in tissues.
    • 4. Like Mb
  9. 1. What is Hb saturation in arterial blood? How about in veins?
    2. Do Hb's alpha and beta subunits interact with one another? Mainly through what types of interactions? (2 major ones, 1 minor)
    3. How do A1 and A2 subunits interact with each other?
    4. How do B subunits interact within each subunit?
    5. Which alpha subunit interacts with which beta unit? How?
    • 1. 96%, 60%.
    • 2. Yes, through noncovalent interactions (ELECTROSTATIC, hydrophobic and H-bonding)
    • 3. A1 and A2 interact with each other through their N and C termini.
    • 4. Through salt bridges formed between His+ and Asp-.
    • 5. A1 and A2's Lys+ forms salt bridge with COO- (C terminus) of each B subunit.
  10. 1. How does Hb respond to environmental changes?
    2. What does the sigmoidal dissociation curve of Hb tell us? (2).
    3. What does cooperativity facilitate?
    1. Hb can switch between a low affinity state (T -tense) and high affinity state (R - relaxed) in different areas of the body. Example, would have high affinity in lungs and low affinity in tissues.

    2. (1) Diagnostic of multiple binding sites and (2) cooperativity/allostericity.

    3. Cooperativity facilitates more sensitive response to concentration of ligand (O2).
  11. 1. How does P50 of Hb compare to P50 of Mb? What does this mean?
    2. How are T state and R state related?
    3. Which state is favored in deoxygenated environment - low PO2 (i.e., tissue)?
    4. Which state is favored in oxygenated environment - high PO2 (i.e., lungs)?
    • 1. Hb's is higher, meaning it has a lower affinity for O2.
    • 2. They are the same molecule (Hb) with conformational changes - they're in equilibrium with each other.
    • 3. T state
    • 4. R state
  12. 1. What is the sequential model and what does it explain?
    2. What does R state's dissociation curve look like?

    1. Ligand binding induces conformational change in an individual subunit --> a similar conformational change in adjacent subunit --> binding of 2nd ligand more likely.

    Explains cooperativity

    2. Like myoglobin's dissociation curve.