Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: Application to the lactose permease of Escherichia coli

John C Voss, Wayne L. Hubbell, H. Ronald Kaback

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metalnitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and >23 Å from the metal binding site. These data are consistent with an α-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Original languageEnglish (US)
Pages (from-to)12300-12303
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number26
DOIs
StatePublished - Dec 19 1995

Keywords

  • electron paramagnetic resonance
  • helix packing
  • transmembrane helices

ASJC Scopus subject areas

  • Genetics
  • General

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