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. 1995 Aug;4(8):1563–1570. doi: 10.1002/pro.5560040814

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

H Wendt 1, E Dürr 1, R M Thomas 1, M Przybylski 1, H R Bosshard 1
PMCID: PMC2143184  PMID: 8520482

Abstract

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.

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Selected References

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  1. Alber T. Structure of the leucine zipper. Curr Opin Genet Dev. 1992 Apr;2(2):205–210. doi: 10.1016/s0959-437x(05)80275-8. [DOI] [PubMed] [Google Scholar]
  2. Baxevanis A. D., Vinson C. R. Interactions of coiled coils in transcription factors: where is the specificity? Curr Opin Genet Dev. 1993 Apr;3(2):278–285. doi: 10.1016/0959-437x(93)90035-n. [DOI] [PubMed] [Google Scholar]
  3. Cohen C., Parry D. A. Alpha-helical coiled coils: more facts and better predictions. Science. 1994 Jan 28;263(5146):488–489. doi: 10.1126/science.8290957. [DOI] [PubMed] [Google Scholar]
  4. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  5. Engel M., Williams R. W., Erickson B. W. Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue alpha-helical dimers. Biochemistry. 1991 Apr 2;30(13):3161–3169. doi: 10.1021/bi00227a002. [DOI] [PubMed] [Google Scholar]
  6. Harbury P. B., Kim P. S., Alber T. Crystal structure of an isoleucine-zipper trimer. Nature. 1994 Sep 1;371(6492):80–83. doi: 10.1038/371080a0. [DOI] [PubMed] [Google Scholar]
  7. Hodges R. S., Semchuk P. D., Taneja A. K., Kay C. M., Parker J. M., Mant C. T. Protein design using model synthetic peptides. Pept Res. 1988 Sep-Oct;1(1):19–30. [PubMed] [Google Scholar]
  8. Hurst H. C. Transcription factors. 1: bZIP proteins. Protein Profile. 1994;1(2):123–168. [PubMed] [Google Scholar]
  9. König P., Richmond T. J. The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J Mol Biol. 1993 Sep 5;233(1):139–154. doi: 10.1006/jmbi.1993.1490. [DOI] [PubMed] [Google Scholar]
  10. Lamb P., McKnight S. L. Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trends Biochem Sci. 1991 Nov;16(11):417–422. doi: 10.1016/0968-0004(91)90167-t. [DOI] [PubMed] [Google Scholar]
  11. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  12. Leder L., Wendt H., Schwab C., Jelesarov I., Bornhauser S., Ackermann F., Bosshard H. R. Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides. Eur J Biochem. 1994 Jan 15;219(1-2):73–81. doi: 10.1111/j.1432-1033.1994.tb19916.x. [DOI] [PubMed] [Google Scholar]
  13. Monera O. D., Zhou N. E., Kay C. M., Hodges R. S. Comparison of antiparallel and parallel two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization. J Biol Chem. 1993 Sep 15;268(26):19218–19227. [PubMed] [Google Scholar]
  14. O'Neil K. T., DeGrado W. F. A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science. 1990 Nov 2;250(4981):646–651. doi: 10.1126/science.2237415. [DOI] [PubMed] [Google Scholar]
  15. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  16. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  17. O'Shea E. K., Rutkowski R., Stafford W. F., 3rd, Kim P. S. Preferential heterodimer formation by isolated leucine zippers from fos and jun. Science. 1989 Aug 11;245(4918):646–648. doi: 10.1126/science.2503872. [DOI] [PubMed] [Google Scholar]
  18. Scholtz J. M., Qian H., York E. J., Stewart J. M., Baldwin R. L. Parameters of helix-coil transition theory for alanine-based peptides of varying chain lengths in water. Biopolymers. 1991 Nov;31(13):1463–1470. doi: 10.1002/bip.360311304. [DOI] [PubMed] [Google Scholar]
  19. Weinmann W., Parker C. E., Baumeister K., Maier C., Tomer K. B., Przybylski M. Capillary electrophoresis combined with 252Cf plasma desorption and electrospray mass spectrometry for the structural characterization of hydrophobic polypeptides using organic solvents. Electrophoresis. 1994 Feb;15(2):228–233. doi: 10.1002/elps.1150150139. [DOI] [PubMed] [Google Scholar]
  20. Zhou N. E., Kay C. M., Hodges R. S. Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil. Biochemistry. 1992 Jun 30;31(25):5739–5746. doi: 10.1021/bi00140a008. [DOI] [PubMed] [Google Scholar]
  21. Zhou N. E., Kay C. M., Hodges R. S. The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability. Protein Eng. 1994 Nov;7(11):1365–1372. doi: 10.1093/protein/7.11.1365. [DOI] [PubMed] [Google Scholar]

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