Hi,

PagP, an integral outer membrane enzyme of e.coli I am working on, has been shown to control the cytoplasmic event of lipopolysaccharide biosynthesis when activated . From the structural details of PagP, it seems that 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.

Your help will be really appreciated.

Thank you in advance

they have superimopsable image with chymotrypsin catalytic triad

 

This is like an aprotic nucleophile enzyme? I would think that this mechanism might be involved in the actual lipopolysaccharide biosynthesis. But I'm not sure if you'll find a putative signal transduction pathway. It sounds sort of structurally advantageous.

 

Anyways this is very complicated. If you alter the metabolic growth medium and the activity changes, I think you can say that it is not a signal transduction pathway. Maybe just the palmitate acid. Or perhaps something which leads to a true metabolic pathway change.

 

Otherwise I think you'd need to use DNA alterations.

Edited May 13, 201312 yr by vampares

Is this a homework question? There are quite a number of suitable experiments. The general elements are the modification of the elements of interest (most cases that would be mutagenesis) and monitoring the outcome (for regulatory elements it could be expression studies, for example).

For protein-protein interactions there are also many techniques available, depending on what you want to see at the end and what kind of instrumentation you may have available. Considering that this may be related to some kind of course you should probably just look at the techniques being discussed to find the suitable ones/