Membrane proteins and impure detergents: procedures to purify membrane proteins to a degree suitable for tryptophan fluorescence spectroscopy.
Investigation of the membrane-embedded mannitol permease of Escherichia coli (EIImtl) steady-state tryptophan fluorescence was hampered by fluorescent impurities arising from detergents and other sources during the isolation. The signals from these impurities could not be distinguished from tryptophan fluorescence on the basis of lifetimes or emission spectra. Consequently, a tryptophan-minus mutant of EIImtl, EIImtl(Trp-), was constructed to address this problem. The findings were that the fluorescent impurities, present in the detergents and/or arising from the action of the detergents on plastic vials and tubing used during the isolation procedure, accumulate in enzyme solutions to levels comparable to the signal from the tryptophan residues in the protein. The high affinity of these impurities for EIImtl makes them impossible to remove by dialysis, by reconstitution of the protein with pure phospholipids, or by detergent exchange when the protein is immobilized on a resin. A procedure was developed to completely remove all fluorescent impurities from the nonionic polyethylene glycol-based detergent, decylpenta(ethylene glycol) (C10E5). This detergent and modified isolation procedures yield EIImtl(Trp-) and single tryptophan mutants in which the impurities no longer interfere with the tryptophan emission signal. The methodologies presented in this paper might make it possible to study the fluorescence of tryptophan residues in other membrane proteins without the interference of impurities with similar fluorescence properties.
Dijkstra
D S
DS
Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, AG Groningen, 9747, The Netherlands.
Broos
J
J
Robillard
G T
GT
D003902:Detergents; D004340:Drug Contamination; D008353:Mannitol / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D003902:Detergents; D008565:Membrane Proteins; D008353:Mannitol; D014364:Tryptophan