Tryptophan replacements in the trp aporepressor from escherichia coli: Probing the equilibrium and kinetic folding models

Authors

  • Craig J. Mann,

    1. Department of Chemistry, The Center for Biomolecular Structure and Function, and The Biotechnology Institute, The Pennsylvania State University, University Park, Pennsylvania 16802
    Current affiliation:
    1. Eli Lilly and Company, Lilly Corporate Center Mail Drop Code 3227, Indianapolis, Indiana 46285
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  • Catherine A. Royer,

    1. School of Pharmacy, University of Wisconsin at Madison, Madison, Wisconsin 53706
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  • C. Robert Matthews

    Corresponding author
    1. Department of Chemistry, The Center for Biomolecular Structure and Function, and The Biotechnology Institute, The Pennsylvania State University, University Park, Pennsylvania 16802
    • Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802
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Abstract

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844–1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011–7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591–597) provides a simple explanation for this phenomenon.

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