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. 1993 Jul;2(7):1072–1084. doi: 10.1002/pro.5560020701

Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis.

J C Hu 1, N E Newell 1, B Tidor 1, R T Sauer 1
PMCID: PMC2142419  PMID: 8102921

Abstract

Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short alpha-helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a lambda repressor-leucine zipper fusion protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and non-functional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix-forming propensity and favorable intrasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.

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Selected References

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