Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Alexej Kedrov, Christine Ziegler, Harald Janovjak, Werner Kühlbrandt, Daniel J. Müller

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71 Citations (Scopus)

Abstract

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane α-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.

Original languageEnglish
Pages (from-to)1143-1152
Number of pages10
JournalJournal of Molecular Biology
Volume340
Issue number5
DOIs
Publication statusPublished - 23 Jul 2004
Externally publishedYes

Keywords

  • AFM, atomic force microscopy
  • BSE, bovine spongiform encephalophaty
  • CJD, Creutzfeldt-Jakob
  • Escherichia coli
  • folding kinetics
  • membrane protein
  • molecular interactions
  • NhaA, sodium-proton antiporter
  • secondary structure elements
  • WLC, worm-like chain

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