Hi all,

I just listened to a discussion about protein domains and a potential substrate binding. A question was given and I was thinking how to tackle the problem. Let's say: a functional protein consists of 2 major domaind. It is assumed that the N-terminal domain is considered to bind the substrate and the C-terminal domain binds to hydrohilic compound and eventually the hydrohilic group is linked with the substrate. As you may have guessed we're talking about a transferase enzyme. The question is how can you proof that the N-terminal domain really binds to the substrate (e.g. any toxic substance). I was thinking to change amino acids in the domain and measure the substrate concentration or the product formation by MS. A colleague of mine suggested doing an EMSA but how can you be sure that it's this domain that does the job. Do you have any other solution how this issue could be solved ?