How to elucidate Signal transduction pathway? - (May/07/2013 )

Hi,

The integral outer membrane enzyme I am working on PagP has been show to control the cytoplasmic event of lipopolysaccharide biosynthesis when activated . From the structural details of PagP, it seems like three amino acid residues in the inner leaflet region are organise in the catalytic triad (They have superimopsable image with chymotrypsin catalytic triad). Now, we want to know whether these three residues are actually working as catalytic triad and doing the signal transduction. What reliable experiments can answer this question?
Since, Outer membrane protein is affecting the cytoplasmic event that means there must be some proteins in periplasm and inner membranes that are co-operating in signal transduction. What experiments can allow us to find out which proteins are participating in this putative signal transduction pathway.

Thank you in advance

YOu can do site-directed mutagenesis to alter the three aminoacids and see if that alters the catalytic ability.

You could do IP of your protein and mass-spectroscopy to identify co-precipitated proteins.

Thank you very much for your response. What if I do the RNA-seq or qPCR to find out the downstream target of PagP mediated signalling? Will be there be any difference seen at the level of gene and protein?

and which is more reliable?

Potentially you can use both as they look at different things in many respects -RNAseq or a genechip could be used to identify potential targets and then qPCR used to confirm.

That means I can do RNA-seq in stead of CO-IP?

No - they give you different answers. RNAseq tells you if you might have a gene transcript present (but says nothing about the protein) and potentially tells you how much is present, whereas the Co-IP tells you if two proteins are actually physically interacting, not just via a pathway.

Oh, that's good to know. Your answers are quite helpful for me in driving my project. Thank you very much.
I have some more queries. Suppose, if gene A was found differentially regulated in the RNA-seq experiment, then how can I established the relationship between my protein (PagP) and the protein coded by gene A biochemically?

DIrect interaction - by co-IP - otherwise you could try knock down of your GOI and see how the other is affected.

Thank you. I am getting greedy here. Regulation can be at the level of gene or transcriptome and sometimes enzymes shows allosteric regulation and can't be detected through gene expression expression experiments. Is that correct? Is there any possibilty of having allosteric regulation of my enzyme (PagP)? What experiments can determine allosteric regulation?