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Conclusions and Future Work - Coggle Diagram
Conclusions and Future Work
Results
Structure of the crosslinker as well as the irradiation affects the crosslinking efficiency
BN doesn’t give crosslinking products while AZ shows the most efficient crosslinking
After 30 min crosslinking efficiency plateues
Crosslinking efficiency of gel experiments don’t match the ligand preferences of the protein which could be due to the size of the ligand-BP but not due to change in ligand affinity when ligand is attached to DNA (NanoBRET confirms this)
purified protein mock selections correlate well with the gel results in terms of the AZ having higher recovery than DZ but doesn’t match in terms of ligand preferences.
cell lysate selections still show higher recovery for the affinity than crosslinking but crosslinking results agree well with the purified protein trends
selection with a mock DEL indicates the prominent effect of stringent washes in terms of getting rid of the noise in the data
this fits well with the claim that even thous the crosslinking would be low, the binders will still be determined since crosslinking allows stringent washes to be performed which gets rid of the non-binders better than mild washes
genetic code expansion of the CAIX resulted in 3 active site mutants and one protein surface mutant
1 out of three of the mutants showed enrichment to the GLCBS-BC3
the same mutant in a mock selection gave convincing results to conclude that the genetic code expansion allowed the crosslinking of the ligand with the protein upon irradiation of the sample
Future Work
CAII can be used in the genetic code expansion
this would increase the amount of protein expression
CAIX can be used to do screenig on live cell membranes
CAIX can be genetic code expanded further to find residues that give positive results to compare the positioning of the DiazK with respect to the ligand
different nnAA can be tried to increase the crosslinking efficiency
such as aryl azide
do selection with a mock DEL using the mutant CAIX
Applications
screening of protein surface for allosteric sites
can be used with unmodified DELs, i.e. the ones that can be purchased
No need for extra work in designing a headpiece that can anneal with the CP
a method of choice if you want to decrease the noise in DEL sequencing