Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters.

Using the example of sodium/proton antiporter from Escherichia coli NhaA, we review the capabilities of single-molecule atomic force microscopy and force spectroscopy to observe structural and functional insights of a membrane protein, which are not attainable by other traditional methods. While atomic force microscopy provides high-resolution topographs of single membrane proteins, their oligomeric state and assembly, single-molecule force spectroscopy experiments detect molecular interactions of the protein. The sensitivity of this method makes it possible to detect and locate interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within them. Controlled refolding experiments using single-molecule force spectroscopy observed individual secondary structure segments folding into the functional protein. Various folding pathways of NhaA were detected, each one exhibiting a certain probability to be taken. Time-lapse refolding experiments enabled determining the folding kinetics and hierarchy of individual secondary structural elements. Recent examples detected and located the ligand binding of an antiporter. Similarly, inhibitor binding and location can be detected which in future may guide towards comparative studies of agonist and antagonist altering the functional state of a membrane protein. We review current and future potentials of these approaches to characterize the action of pharmacological molecules on the antiporter function.