2nd Exam Biochem 104b

Write down your name on this exam sheet and the following pages (graphing paper andpicture page) and hand them in with the exam.

Important!!!!!:- You must answer both question 1 and 3.

- You can choose between answering question 2a (secondary structure) or 2b(tertiary structure).

- If you answer both 2a and 2b I will count only the question which has thebetter score (e.g. 2a=25 points 2b=16 points -> your score for question 2 =25points)

Question 1) Primary structure (30 points)

a) Draw the backbone of a tri-peptide indicating the side chains as “R”.

b) Indicate the bonds that define the phi and psi torsion angles.

c) Draw a cis and a trans peptide bond. Why are trans peptide bonds so much morecommon in general? Which amino acid is the exception to this rule and why?

Question 2a) Secondary Structure (30 points)

a) What are the characteristic features that identify an alpha helix? (e.g number ofresidues per turn, where do the side chains point, what is the hydrogen-bondingpattern etc etc.)

b) Using the criteria you identified above, which of the 5 poly-alanine helices on thepicture page are real alpha helices? For those helices that are not real alpha helicesidentify the features that make them “wrong”. (e.g. D- instead of L- amino acids,too many or too few residues per turn etc.)

HINT: There may be more than one real alpha helix.

c) Alpha helices have an electrostatic dipole moment, but beta sheets do not, explainwhy.

Question 2b) Tertiary structure (30 points)The picture page shows three views of a ribbon diagram of a protein.

a) If you would look at the primary sequence of this protein, what pattern of aminoacid properties would you expect for the beta sheet regions and why?

b) Draw a topology diagram of this protein. Label the secondary structure elements(alpha1, beta1. alpha2 etc.)

c) Imagine you were a protein engineer, and you would want to design a smallerversion of this protein, but want to preserve its structural integrity. Which regionsof the protein do you think you could eliminate? Circle them on the ribbondiagrams.

Question 3) Protein melting curves (40 points)

Your lab is investigating a receptor molecule and wants to use your new physicalchemistry skills to answer some new questions. Here is the state of the project when youcome in:

- The lab has designed a mutant of the receptor, which converts an Asp in aflexible, solvent-exposed loop on the protein surface into a Phe.

- They determined the structures of both the mutant and the wild-type protein andexcept for the side-chain change the two structures look identical.

- Protein stability experiments on the wild-type protein gave: DHunfold=32kcalDSunfold=90cal

You get the following data from a protein melting experiment of the mutant

Temp in deg C rel. fluorescence intensity25 0.540 0.6155 1.1570 1.8085 2.0

NOTE: Protein stability experiments on the wild-type protein gave: DHunfold=32kcalDSunfold=90cal

a) Determine the Tm, DHunfold, DSunfold, DGunfold (at 20 deg.C) of the mutant and theDDHunfold, DDSunfold, DDGunfold (at 20 deg C) relative to the wild-type protein.

b) Somebody in your lab wonders if the unfolded form of the mutant protein may bepopulated enough to play a biological role in the signaling reaction. To help out,calculate the fraction of unfolded molecules at 20 degrees C. Why would it be

hard to measure the fraction of unfolded mutant protein at 20 deg. Cexperimentally?

c) Now it is head scratching time! Do the results from the mutant surprise you?What effect, if any, would you have expected to see for the mutation? How couldyou rationalize the result. Two hints: i) remember that DG is defined by two statesof the protein. Which of the two states may be responsible for the effect of themutant. ii) Remember what you learned about the molecular nature of thehydrophobic effect?

FIGURES for Question 2a and 2b