Discussion Problems BT 3016

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?

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

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?

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

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?

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

Which will separate? M r (subunit) subunitspI pH 8 A14,200 Monomer B13,900 monomer C67,000 monomer D66,950 dimer

By the following techniques?

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?

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?

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

Measure initial rates at several values of [S] 0 How do you find K M and V max [S] 0 =

Find Initial Rate for each [S] 0 Slope (convert to mM/min)

Make table for all [S] 0,V 0 [S] 0 V0V /[S] 0 1/V

Plot 1/V 0 vs. 1/[S] 0 1/Vmax -1/K M

Cooperativity? V0V0 [S] 0.9V max.1V max V max [S].9Vmax [S].1Vmax [S].9Vmax

Reversible Inhibition 1/V o 1/S o ? competitive Inhibition [I] 1/V o 1/S o ?competitive Inhibition [I] 1/V o 1/S o ?competitive Inhibition [I] -1/K M 1/V max X I = X (1 + [I]/K I )

Effect of pH? EH 2 + S EH 2 S EH 2 + P E -2 + S ES -2 E -2 + P EH - + S EHS - EH - + P +H +

Effect of T? Choose a good pH Measure V 0 over a pH range Plot what? V 0 vs T ln V 0 vs. 1/T ln (V 0 /T) vs. 1/T