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

Fingerprint

Escherichia
metal ions
Labeling
lactose
Metal ions
binding sites
Metals
Binding Sites
metals
Ions
membrane proteins
Membrane Proteins
Spin Labels
hounds
mutants
Biochemistry
avidin
Avidin
electron paramagnetic resonance spectroscopy
biotin

ASJC Scopus subject areas

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

Cite this

Helix packing in the lactose permease of escherichia coti as determined by use of designed metal ion binding sites and site-directed gpin labeling. / Voss, John C; Hubbell, Wayne L.; Ronald Kaback, H.

In: FASEB Journal, Vol. 10, No. 3, 1996.

Research output: Contribution to journalArticle

@article{64ff835b0cf94379aa0189b9b9c6ce65,
title = "Helix packing in the lactose permease of escherichia coti as determined by use of designed metal ion binding sites and site-directed gpin labeling",
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 {\AA}. 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.",
author = "Voss, {John C} and Hubbell, {Wayne L.} and {Ronald Kaback}, H.",
year = "1996",
language = "English (US)",
volume = "10",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "3",

}

TY - JOUR

T1 - Helix packing in the lactose permease of escherichia coti as determined by use of designed metal ion binding sites and site-directed gpin labeling

AU - Voss, John C

AU - Hubbell, Wayne L.

AU - Ronald Kaback, H.

PY - 1996

Y1 - 1996

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33748969531&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748969531&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:33748969531

VL - 10

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 3

ER -