Probing folded and unfolded states of outer membrane protein a with steady-state and time-resolved tryptophan fluorescence.
Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.
Kim
Judy E
JE
Beckman Institute and Department of Chemistry, California Institute of Technology, Pasadena, California 91125, USA.
Arjara
Gitrada
G
Richards
John H
JH
Gray
Harry B
HB
Winkler
Jay R
JR
D016880:Anisotropy; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001703:Biophysics / Q000379:methods*; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D008968:Molecular Conformation; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D008823:Micelles; C078167:OMPA outer membrane proteins; D014364:Tryptophan; D014508:Urea; D004134:Dimyristoylphosphatidylcholine