Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding.
The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.
Mann
C J
CJ
Department of Chemistry, Pennsylvania State University, University Park 16802.
Matthews
C R
CR
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D012097:Repressor Proteins; C027190:TRPR protein, E coli; C027132:1-anilino-8-naphthalenesulfonate