Mechanisms of folding and misfolding of membrane proteins are of interest in cell biology. Recently, we have established single-molecule force spectroscopy to observe directly the stepwise folding of the Na +/H+ antiporter NhaA from Escherichia coli in vitro. Here, we improved this approach significantly to track the folding intermediates of a single NhaA polypeptide forming structural segments such as the Na +-binding site, transmembrane α-helices, and helical pairs. The folding rates of structural segments ranged from 0.31 s-1 to 47 s-1, providing detailed insight into a distinct folding hierarchy of an unfolded polypeptide into the native membrane protein structure. In some cases, however, the folding chain formed stable and kinetically trapped non-native structures, which could be assigned to misfolding events of the antiporter.
- Atomic force microscopy
- Folding kinetics
- Molecular interactions
- Single-molecule force spectroscopy
- Sodium/proton antiporter