Helix packing in the lactose permease of escherichia coti as determined by use of designed metal ion binding sites and site-directed gpin labeling

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

Research output: Contribution to journalArticle

Abstract

As shown previously [Voss, J., Salwinski, L,. Kahack, H. R & Hubbell, W. L., P.N.A.S. USA (J995) 12295-99; Voss. J., Kaback, H. R. & Hubbell. W. L..P.N.A.S. USA (1995) 12300-3], the magnetic dipolar interaction between site-directed metal/nitroxide pairs can be exploited to measure distances in proteins. This is a powerful method for genera! use. part cularly with membrane proteins that are difficult to crystallize. Using the metal-spin label approach, both a paramagnetic metal ion binding site and a nitroxide side-chain were introduced at selected positions in the lactose permease of E. coli, a paradigm for polytopic membrane proteins. Thus, individual Cys residues were introduced into a lactose permease mutant devoid of native Cys residues for site-directed spin-labeling. In addition, the mutants were engineered to contain a high-affinity divalent metal ion binding site in a trunsmembrane domain of the protein and a biotin domain fio avidin-affinity purification [He, M. M., Voss. J., Hubbell. W. L. & Kaback, H. R. (1995) Biochemistry 34, 15667-70]. In the presence of Cu( II) the electron paramagnetic resonance spectrum of a nitroxide attached to Cys-234 on transmembrane helix VII is broadened due to dipolar interactions with the hound metal. Based on the spectral changes, the distance between the two paramagnetic centers was calculated at 18 Å. Together with measurements involving neighboring residues 233, 235 and 236, the distance and orientation of helix VII with respect to the bound melal is presented.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number3
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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