MBB 400a/700a
Problem Set #3

1. A) What is fluorescence energy transfer and why does it occur?
B) For which of these distances would it be suitable to do experiments utilizing fluorescence energy transfer?  0.1 nm, 10 nm, 100 nm, 1000 nm
C) How can you monitor whether fluorescence energy transfer is occurring?
D) Which fluorescent probe(s) might serve as an energy donor to the probe FITC? (Consult the table from the notes handed out in class on 9/15/98.)  Identify another possible donor/acceptor pair from this list.

2. Visit the website at:
      http://www.embl-heidelberg.de/Services/serrano/agadir/agadir-start.html
Read the description and instructions for use.
You are asked to mutate the following polypeptide so its helical propensity is increased from 56% to above 80%.
RPDLEADRELLREVLELMKRLQEVQTEDNDV
Once at the website, click “Submit to agadir.”  Type in the sequence above and hit next page.  You may submit the next two parameter screens with the default values.  Finally hit “Run Calculation” then “output.”  The helical propensity for the peptide will be displayed.   You can then change whatever residues from the above sequence that you wish and repeat the process to see if you have increased the helical propensity with your mutations.  Try to make rational mutations using what you have learned from Lynne’s lectures.  Do not change the length of the sequence and also try to keep the number of mutations to a minimum.  Also, don’t change half of the residues to alanine – this is not what I’m looking for here.  When you have succeeded, email me your answer at: jennifer.larson@yale.edu
Also include a short paragraph explaining the rationale behind some of your mutations.  (You may want to keep track of each mutation that shows improvement by keeping it in a word processing program.  If you mess the whole thing up with a bum mutation, you can go back to the previous sequence instead of starting all over.)

3. Read the Weissman, et al., 1997, paper that accompanies this problem set before answering the following questions:
A) In simple terms, explain what is being measured in Figure 1.  What do changes in anisotropy tell us?
B) Why is GFP a convenient substrate to measure protein folding in this paper?  How do we know that we know that the folded GFP is not tumbling loose in the SR1 cavity?  What control was done to show that the GFP is hindered when bound by SR1 (relative to GFP by itself in solution)?
Don’t worry about experiments that we haven’t covered in class yet.  Try to get as much out of the paper as you can.
 
4. Sketch what the CD spectrum would look like for polypeptide in an ?-helix conformation.  You ask a friend at the Keck peptide synthesis facility (at the med school) to synthesize this peptide for you.  He is new on the job and foolishly makes your peptide entirely from D-amino acids.  What would the CD spectrum look like now?