Discussion Problems

1
Discussion Problems

• BT 3016

2
Protein Structure

• How do the amino acids differ from one another
  structurally?
• What properties differ?
• What are the kinds of secondary structure?
  Describe them.
• What is quaternary structure?
• What causes proteins to fold?

3
Structure - continued

• What is a Ramachandran diagram?
• What are the four classifications of protein
  folds?
• How can you identify a transmembrane protein?

4
pH, pKa, HH equation

• What is the charge on an amino acid in a solution
  that is 0.1 unit below its pI?
• How do you make a buffer of a particular pH and
  concentration?
• 50mM glycine buffer, pH 8.8
• What is its ionic strength?

5
Solubility

• Describe three ways to cause a protein to
  precipitate?
• Describe three ways to cause an insoluble protein
  to dissolve?

6
Separations

• What techniques separate according to the size of
  a protein?
• What techniques separate according to charge on a
  protein?
• What techniques separate based on a combination
  of both charge and size?

7
Separation by Size

• What fundamental difference is there between
  determination of molecular weight by SDS PAGE and
  by Size Exclusion chromatography?

8
Which will separate?
Mr (subunit) subunits pI Charge _at_ pH 8
A 14,200 Monomer 9.5 14
B 13,900 monomer 5.6 -4
C 67,000 monomer 5.2 -8
D 66,950 dimer 4.9 -20
9
By the following techniques?
10
Activity

• What is an activity unit?
• How is it different from a rate?
• What is total activity?
• What is specific activity?
• What is purification fold?
• What is recovery?

11
Reaction Order

• Define the order of the reaction
• Define the order in a component
• What does pseudo zero order mean?
• How could a reaction be pseudo zero order in a
  component?
• What does the order of reaction imply about a
  mechanism?

12
Determine the order of the reaction

• Initial rates
• Change one reactant
• Measure initial rate
• See how the rate changes
• Integrated Rate Equation
• Follow the reaction until significant changes in
  concentrations occur
• See which integrated rate equation describe the
  data

13
How do you find KM and Vmax
Measure initial rates at several values of S0
14
Find Initial Rate for each S0
15
Make table for all S0,V0
S0 V0
5 83
10 143
20 222
40 308
80 380
1/S0 1/V0
200 0.012
100 0.007
50 0.0045
25 0.0033
12.5 0.0026
16
Plot 1/V0 vs. 1/S0
17
Cooperativity?
18
Reversible Inhibition
I
1/Vmax
? competitive Inhibition
-1/KM
I
1/Vo
1/Vmax
?competitive Inhibition
XI X (1 I/KI)
1/So
-1/KM
19
Effect of pH?
20
Effect of T?

• Choose a good pH
• Measure V0 over a pH range
• Plot what?
• V0 vs T
• ln V0 vs. 1/T
• ln (V0/T) vs. 1/T