Abstract
Specific side-by-side interactions between transmembrane α-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane α-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane α-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the α-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7683-7689 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 267 |
| Issue number | 11 |
| State | Published - Jan 1 1992 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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Glycophorin A dimerization is driven by specific interactions between transmembrane α-helices. / Lemmon, M. A.; Flanagan, J. M.; Hunt, J. F.; Adair, B. D.; Bormann, B. J.; Dempsey, C. E.; Engelman, D. M.
In: Journal of Biological Chemistry, Vol. 267, No. 11, 01.01.1992, p. 7683-7689.Research output: Contribution to journal › Article
TY - JOUR
T1 - Glycophorin A dimerization is driven by specific interactions between transmembrane α-helices
AU - Lemmon, M. A.
AU - Flanagan, J. M.
AU - Hunt, J. F.
AU - Adair, B. D.
AU - Bormann, B. J.
AU - Dempsey, C. E.
AU - Engelman, D. M.
PY - 1992/1/1
Y1 - 1992/1/1
N2 - Specific side-by-side interactions between transmembrane α-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane α-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane α-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the α-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.
AB - Specific side-by-side interactions between transmembrane α-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane α-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane α-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the α-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.
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UR - http://www.scopus.com/inward/citedby.url?scp=0026686793&partnerID=8YFLogxK
M3 - Article
C2 - 1560003
AN - SCOPUS:0026686793
VL - 267
SP - 7683
EP - 7689
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 11
ER -