Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate.
Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)
Gardner
J A
JA
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
Matthews
K S
KS
D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D007785:Lactose / Q000737:chemistry*; D009154:Mutation; D009282:Naphthalenesulfonates / Q000737:chemistry*; D010449:Peptide Mapping; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D009282:Naphthalenesulfonates; D012097:Repressor Proteins; C006421:1,5-I-AEDANS; D007785:Lactose