TRYPTOPHAN REPLACEMENTS IN THE TRP APOREPRESSOR FROM ESCHERICHIA-COLI- PROBING THE EQUILIBRIUM AND KINETIC FOLDING MODELS

Mutants of the dimeric Escherichia coli trp aporepressor are construct ed by replacement of the two tryptophan residues in each subunit in or der to assess the effects on equilibrium and kinetic fluorescence prop erties of the folding reaction. The three kinetic phases detected by i ntrinsic tryptophan fluorescence in refolding of the wild-type aporepr essor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to glo bal rather than local conformational changes. Comparison of equilibriu m fluorescence (Royer, C.A., Mann, C. J., & Matthews, C.R., 1993, Prot ein Sci. 2, 1844-1852) and circular dichroism transition curves induce d 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 interpret ed in terms of enhancement in the population of a monomeric folding in termediate in which the lone tryptophan residue is highly exposed to s olvent, 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 explan ation for this phenomenon.