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.
6 thoughts on “Mapping the domains of full-length huntingtin – next installment of mass spectrometry data”
Rachel, are you surprised that the caspase cleavage region (~500-600aa) isn’t represented by any trypsin or chymotrypsin fragments? It seems one of the only un-cleaved regions that lines up across both proteases. Does this make any testable predictions about the accessibility of this region?
Hi Jeff. Thanks for your comment. The fact that this region (500-600) is the caspase cleavage region already suggests that it is more proteolytically accessible so likely to be cut at multiple points by the digesting protease in the limited proteolysis step of these experiments. Caspases generally recognise specific protein sequence tracts I believe rather than 3D motifs indicating the cleavage sites are in less ordered regions of the protein structure. I would not expect to find a fragment corresponding to 500-600 but perhaps N-term up to 500 and 600 onwards, also in accordance with the domain structure prediction from InterPro. It is interesting that fragments crossing through this region are detected – 100-627&445-1184 for trypsin and 438-1183 for chymotrypsin. However, these fragments are from the 30 minute time point of the limited proteolysis so would perhaps not be detected at a later time point. Does this answer your point? Thanks for commenting! I believe you are my first scientist commenter, how exciting.
Interesting – and you’re right, as far as I know caspases cleave ~tetrapeptide recognition sequences so I guess that part of the protein would have to be pretty accessible for those sites to be found.
The main limitation with these conclusions is that the caspase, or the proteases I am using in my experiments, can act on extended loop regions which protrude from the more folded regions of the huntingtin structure so there may not be a clear break in the overall fold. No way to tell though until we have some higher resolution structural data.
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Thanks for taking the time to comment. I appreciate your feedback. I hope that you continue to follow the work along. Have a great day 🙂