Over the past couple of days I have been continuing to work on mapping the domains of huntingtin, an important thread to this structure-function project. Whilst the modelling data I posted on Monday is super exciting to look at, protein models are very aesthetically pleasing to an X-ray crystallographer such as myself, they are just that, models, and experimental data is always better. So I was delighted when Paul Taylor at SPARC, Sick Kids Hospital, Toronto, returned some new data to me to continue my domain mapping investigations.
The data Paul sent is from an experiment which is essentially a repeat of the last limited proteolysis experiment with tandem mass spectrometry. The key difference is that this time, he digested the gel bands of proteolytically stable huntingtin (the exact same ones as in the previous experiment) with chymotrypsin instead of trypsin. This enzyme has a different specificity for where it will cut in the protein sequence; chymotrypsin after bulky hydrophobic residues, trypsin after positively charged residues. This generates a different series of peptides to be analysed by the mass spectrometer and so can potentially increase the coverage of the regions of protein we are interested in. This is particularly useful when trying to more precisely define the putative domain boundaries of huntingtin as well as acting as a replicate experiment. You can download the raw data files, methods and all of my analysis from Zenodo but you can see a brief summary below:
Pulling all of this data together has given me a better handle on where the domains are likely to be, as well as an idea of how I can potentially design expression constructs of these fragments of huntingtin which would hopefully allow expression and purification of these smaller pieces of protein. I am now beginning to work on the construct design so I will have that for you soon.
If you have any questions, queries or criticisms about this work, please do not hesitate to get in touch.