We have purified our protein of interest, and have shown that FAD is bound, by UV-VIS spectroscopy. We do not understand the role of the FAD binding. I would like to make mutations that disrupt FAD binding, however I do not know which mutations to make! Once these mutations were introduced, we would do in vivo and in vitro studies to probe the purpose of the bound FAD. Does anyone know a good way to determine the FAD binding site? We've considered so far HD exchange, but I am looking for better alternatives.
Hello,
Just a thought. Do you know the amino acid sequence of the protein? In that case you might be able to determine FAD binding domain boundaries using ThreaDom and the possible FAD binding sites using Pfam or some other sort of alignment. FAD binding is very prevalent (at least for the protein that I was studying) so finding the binding site by alignment would not be that complicated.
Yes, we know the amino acid sequence. I will try to use the ThreaDom program. I never heard of that before, so thank you!
The program you want is probably COFACTOR or COACH(preferred) from the same lab, not THREADOM. COACH is a consensus method that includes COFACTOR,FINDSITE, and CONCAVITY
http://zhanglab.ccmb.med.umich.edu/COFACTOR/
http://zhanglab.ccmb.med.umich.edu/COACH/
Actually since you don't have the structure you need to predict it first. Run the I-TASSER program, which will automatically also run the COFACTOR program to predict the binding site
http://zhanglab.ccmb.med.umich.edu/I-TASSER/
Note that it is more difficult to predict the structure of a deeply buried cofactor like FAD then a enzyme whose substrate binds at the surface.
I dont know of an experimental method short of solving the structure besides systematic mutation or doing a NMR backbone assignment and doing an HSQC titration
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