mind blogging question - (Dec/03/2007 )
Hi all,
this is the mind blogger question that has put me in a fix,
protein A interacts with protein B.
now to study the biological function of protein A, i am trying to make a mutant of protein A that does not bind to B but still can carry out the biological
activity of protein B?
what if i dont find such a mutant? 
how else can i study the biological function of protein A interaction with B.
Any suggestions?
syc06
hi,
does not bind to B but still can carry out the biological
activity of protein B?
is B activity is depends on A binding properties?
is B alone active in first instances?
can u explain bit clearly?
sravan.
[quote name='donot lie for ever' date='Dec 4 2007, 05:50 AM' post='118864']
hi all,
i think i should elaborate.
protein A is a repressor, protein B is a activator, they both interact
as a result the genes activated by protein B are repressed by A
now, the promoter regions of genes activated by B have no binding site for protein A,
so, just by interacting with protein B, protein A mediates its repressor activity.
thus, if now i want to find out a mutant of protein B that can still activate the genes activated by
the wild type protein B and can still interact with A, then there is a possibility that i might
be able to define the biological significance of this interaction.
what if i am unable to find such mutant, what other approach can i use
i am tired of thinking about this approach, any good suggestion???
syc06
thus, if now i want to find out a mutant of protein B that can still activate the genes activated by
the wild type protein B and can still interact with A, then there is a possibility that i might
be able to define the biological significance of this interaction.
hi,
from above statement.....
if u want to have a mutant B protein which still binds to promoter and activates similarly like native B and still it can interact with protein A.
in this case it is evidant that this mutation does not have any biological relavance right?
did i understand correctly or am i confused?
sorry, my typo, it shud be cannot interact with protein A but can still activate its target genes, then i have two possibilities:
1. if the genes activated by protein B are still getting repressed on addition of protein A, there is some other binding site or other mechanism by which prot. A
mediates its repressor effect
2. if the genes activated by prot. B are not being repressed on addition of prot.A, then the repression is by binding to prot. B
so, everything is based on finding such mutant, what if i dont find one?
hi all,
i think i should elaborate.
protein A is a repressor, protein B is a activator, they both interact
as a result the genes activated by protein B are repressed by A
now, the promoter regions of genes activated by B have no binding site for protein A,
so, just by interacting with protein B, protein A mediates its repressor activity.
thus, if now i want to find out a mutant of protein B that can still activate the genes activated by
the wild type protein B and cannot still interact with A, then there is a possibility that i might
be able to define the biological significance of this interaction.
what if i am unable to find such mutant, what other approach can i use
i am tired of thinking about this approach, any good suggestion???
syc06
hi,
first study the binding confirmations of both A and B and find out the binding (interacting) domains.
1,
-use truncated protein B (tB), and see whether u can restore the native functions of B (first screening).then if u want u can go for point mutations (second screening).
-once u restore the functions of tB then findout repression capacity of A upon tB binding n gene activation.
-other hand u can use truncated protein A,
find out which domain is important for binding to protein B
find out the catalytic site on protein A
proceed with mutation study to narrow down the search.
for each described step u need to find right experiments to prove the concept.
2,
otherwise u can use antibodies to different epitopes of protein A and B if available. observe their interactions in the presence or abscence of Ab. from their u can find some hints to find the biological relavance.
i hope these ideas will help u to certain extent.
gud luk
sravan.