To keep this question short, let's say I have a novel membrane protein with the known structural fold of a known transporter superfamily so I know its function is a transporter of cellular metabolites (Coenzyme-A, PLP) etc.
Also, say I have computationally docked Coenzyme-A and PLP to the binding site and the poses all have similar negative predicted binding affinity energies etc.
How would you experimentally test or validate that one of these molecules may be transported by the protein?
I am from a bioinformatics background with absolutely no biochemistry lab experience or knowledge, so any insights would be most appreciated, thanks.
The gold-standard method is to reconstitute transport of the substance by the purified protein incorporated into a membrane, usually a liposome. Liposomes can be made to have an inner aqueous compartment that is separated from the outer aqueous environment.
As an example of the experiment, you can add a radiolabeled version of the ligand to proteoliposomes reconstituted with the purified transporter, and add whatever else is needed, such as ATP or an ion gradient across the membrane. Accumulation in the liposomes of the radiolabeled ligand can be measured by separating the liposomes from the external medium and measuring the included radioactivity.
Of course the orientation of the transporter in the liposome can be an important consideration, as well as the direction of transport, and possibly the type of lipids used to make the liposomes.
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