Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli.
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 alpha-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.
Kedrov
Alexej
A
Center of Biotechnology, University of Technology, 01307 Dresden, Germany.
Janovjak
Harald
H
Ziegler
Christine
C
Kuhlbrandt
Werner
W
Muller
Daniel J
DJ
D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers