NCEH1 part-II - assigned 11/2/11, due 11/30/11

Based on our class discussion, where we hopefully decided which modeling approach was best for NCEH1 in part-I, you are now to use the result of that homology modeling to complete part-II.   Just a reminder, the NCEH1  protein sequence  has the NCBI accession codes: Q6PIU2 or gi: 74737782. Go to the UniProt website to get information about this protein useful for this project.

websites which will be of assistance:
Swiss Model  - http://swissmodel.expasy.org/
NCBI PubMed - http://www.ncbi.nlm.nih.gov/sites/entrez/
UniProt - http://www.uniprot.org/
PyMol - http://pymol.org
Protein Data Bank - www.pdb.org
ExPASy  - http://us.expasy.org/
CLUSTALW - http://www.ebi.ac.uk/clustalw/
BOXSHADE  -  http://www.ch.embnet.org/software/BOX_form.html

Please complete the 6 parts below by our class on 11/30/11

1) You are to design a truncated version of human NCEH1 based on the best model from part-I (as discussed in class).  The purpose of making this construct is to mimic the template used in Part-I. It is known that this template was well behaved, which means that the protein was amenable to expression, purification, cyrstallization and solving the structure.   We will hypothesize that if we mimic (through careful truncation) this template, then we'll be able to express a soluble form of NCEH1 that, will likewise be well behaved, and allow us to solve its structure.  Explain your choice of where you will truncate.

2) Redo a ClustalW/Boxshade diagram where you focus in on only NCEH1 versus the single template used.  In your alignment identify the catalytic trial (Ser, His, Asp) as well as regions you plan to truncate.  Also indicate the predicted glycosylation sites (question 5).

3) Design PCR primers which will allow you to truncate and subclone NCEH1 into a pGEX vector so that it expresses as a fusion protein with GST and a thrombin protease site.  In addition to writing the sequence of the primers down, depict (hand drawn  is OK here) how each primer works to bring the truncated DNA sequence into the expression vector.  This is tricky if you haven't done it before and consider this part a group effort.  

Now for a few more functional questions 

4) The protein has a membrane spanning region.  From web based research into this region for NCEH1 explain what the range of residues of this region and any other predicted membrane associated regions of NCEH1.  Draw a crude cartoon of how these regions interact with a membrane that is consistent with what is known about the enzymatic function of this protein.

5) The protein is thought to have glycosylation sites.  Use internet resources to identify the residues likely to be glycosylated and describe how a careful characterization of the role of these sites (in terms of activity, stability, folding) is critical to forming protein crystals to solve the protein's structure.

6) The enzyme NCEH1 is also known as KIAA1363.  What is the alternate physiological reaction that KIAA1363 is believed to catalyze?  Also, describe how inhibitors of KIAA1363 have been identified.  For each of these questions, cite appropriate references of the literature in your answer.   If you were ultimately successful to over-express a truncated form of KIAA1363, which was soluble and well behaved, how could this improve the search for active site competitive inhibitors?

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