Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.
Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)
Royer
C A
CA
School of Pharmacy, University of Wisconsin at Madison 53706.
Mann
C J
CJ
Matthews
C R
CR
D001059:Apoproteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014508:Urea