Observing Folding Pathways and Kinetics of a Single Sodium-proton Antiporter from Escherichia coli

• Published in: Journal of molecular biology 2006-01, Vol.355 (1), p.2-8
• Main Authors: Kedrov, Alexej, Janovjak, Harald, Ziegler, Christine, Kuhlbrandt, Werner, Muller, Daniel J.
• Format: Article
• Language: English
• Subjects: atomic force microscopy; Binding Sites; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; folding kinetics; Kinetics; Microscopy, Atomic Force; molecular interactions; Protein Folding; Protein Structure, Secondary; single-molecule force spectroscopy; Sodium-Hydrogen Exchangers - chemistry; Sodium-Hydrogen Exchangers - metabolism; sodium/proton antiporter
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2005.10.028

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.