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The role of position a in determining the stability and oligomerization state of α-helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins

Published online by Cambridge University Press:  25 April 2001

KURT WAGSCHAL
Affiliation:
Department of Biochemistry and the Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
BRIAN TRIPET
Affiliation:
Department of Biochemistry and the Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
PIERRE LAVIGNE
Affiliation:
Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
COLIN MANT
Affiliation:
Department of Biochemistry and the Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
ROBERT S. HODGES
Affiliation:
Department of Biochemistry and the Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
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Abstract

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 μM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.

Type
Research Article
Copyright
© 1999 The Protein Society

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