Abstract
The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an α-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 211-216 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 37 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 6 1998 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Helix packing in the lactose permease determined by metal-nitroxide interaction. / Voss, John C; Hubbell, Wayne L.; Kaback, H. Ronald.
In: Biochemistry, Vol. 37, No. 1, 06.01.1998, p. 211-216.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Helix packing in the lactose permease determined by metal-nitroxide interaction
AU - Voss, John C
AU - Hubbell, Wayne L.
AU - Kaback, H. Ronald
PY - 1998/1/6
Y1 - 1998/1/6
N2 - The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an α-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.
AB - The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an α-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.
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UR - http://www.scopus.com/inward/citedby.url?scp=0032488624&partnerID=8YFLogxK
U2 - 10.1021/bi972152l
DO - 10.1021/bi972152l
M3 - Article
C2 - 9425041
AN - SCOPUS:0032488624
VL - 37
SP - 211
EP - 216
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 1
ER -