Unfinished project

 

I am often wondering how others are dealing with their unpublished data. Do you have a drawer full of unfinished projects which will never see the light of day? There are a number of drivers that may cause projects to be stopped before completion: Unfunded, priorities changes, data are not worth the additional energy to convert them into a publication, wrong hypothesis,……

 

Here is the story of a project I have abandoned.

 

When I came back in France after my post-doc (a long time ago), one of my project seeks to define the role of sumoylation (an ubiquitin-related post-translational modification) in controlling the functional properties of proteins focusing on the possible structural and dynamic consequences of this modification. Did sumoylation act as structurally independent docking module rather than through the induction of a conformational change in the modified protein?

One obvious target, at least for me, was the transcriptional co-repressor CtBP. It was my post-doc favorite protein and CtBP was known to be sumoylated (ref 1) within its unstructured C-terminal domain (ref 2).

To promote the recombinant expression of SUMOylated proteins in bacteria, we decided to transfer the complete set of enzymes essential for this post translational modification (E1 and E2 enzymes + SUMO-1) to E. coli based on previous work (ref 3). Separating the sumoylated protein from the undesired unmodified fraction (you seldom obtain 100% modification) is often technically challenging so we decided to introduce a 6xHis tag SUMO-1 (ref 4). This strategy did work for IĸBα (ref 4) but I have never been able to optimize the protocol for CtBP. We did obtain roughly 50% of modified and 50% of unmodified CtBP (see the figure below) but because CtBP dimerizes/multimerizes, the separation of the modified from the unmodified substrate in this case was unsuccessful (a single peak in gel filtration).

CTBPS1

 

As the saying goes, every cloud has a silver lining. At the time, I was very positive thinking that the structural study was still possible and even more exciting if I could obtain the structure of a CtBP dimer, one unmodified monomer and one modified monomer. Ah, the foolishness of youth.

 

Frustration, Frustration

So it was time to set up crystallization screenings and we did obtain some exciting crystal ‘hits’. In crystallography, salt crystal is frustration, crystal optimization is frustration, and no diffraction is frustration. CtBP may have a high degree of structural instability in its C-terminus, one modified and one unmodified monomer may lead to sample heterogeneity, whatever but unfortunately NO diffraction. So after a couple of tries (additives, drop ratios, other conditions), I have finally abandoned this project.

affinage ctbp1 E

Maybe I did not try hard enough, maybe one day someone will solve the structure of sumoylated CtBP or maybe one day, I will try again.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google+ photo

You are commenting using your Google+ account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

w

Connecting to %s