Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate.
A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding [Shao, X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-9949]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms. The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts. Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure. When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.
Shao
X
X
Department of Chemistry, Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park 16802, USA.
Matthews
C R
CR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D019281:Dimerization; D005454:Fluorescence Polarization; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D010649:Phenylalanine; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins