Fundamentals of Sequence Analysis, 1998-1999

Problem set 6: Protein tools.

If you get stuck, refer to the OpenVMS and GCG resources in the class home page.


Sequence data used in this problem set, for offsite readers:

Problem group 1. Physical properties

You have isolated the cDNA for a protein that interests you. The predicted translation is present in CLASS:PROTEIN1.PEP.

1A. You want to determine if the protein is likely present in a subcellular fraction, which you have run on a 2D gel (SDS-PAGE/isoelectric focusing.) There are spots at:

Spot   MW (kd)    Isoelectric pH
 A      8.1        6.5
 B     10.7       10.3
 C     22.3        9.6
 D     47.2        5.4
 ( plus an assortment of pH and MW markers)

If it is there, which one is it?

1B. You want to further confirm that the spot you see is actually the protein that you have isolated. You run a preparatory isoelectric focusing gel and isolate the band at pH 10.3, then digest it with CNBr and run the fragments out on an SDS-PAGE gel. THEN you calculate what the expected fragment sizes should have been. What happens?

Problem group 2. What the heck is it?

After a bit more work you have isolated a second protein from this complex. One pass through the protein sequencer yielded this sequence: YAACSTPQ

2A. Design a primer that will be radioactively labeled and used to screen a rat cDNA library for this sequence.

You have sequenced the cDNA clone and found an open reading frame that translates to CLASS:PROTEIN2.PEP. Assume that a database search failed to turn up any similar sequences (don't try it - this example sequence is made from bits and pieces of several proteins and will actually return hits.)

2B. What can you say about this protein?

Problem group 3. Treading on thin ice - what is the secondary structure?

A third protein from this complex has been isolated and fully sequenced. The sequence is stored in the file CLASS:PROTEIN3.PEP, A series of mutations in this protein have been isolated, and all fall between amino acids 100 and 200.

3A. What is the secondary structure in that region?

3B. Suppose that replacing any of the amino acids between 171 and 175 with a proline radically decreases the function of this enzyme. Propose a model based on the secondary structure predictions to explain this.