title: Lipid binding of the exchangeable apolipoprotein apolipophorin III induces major changes in fluorescence properties of tryptophans 115 and 130.
abstract: The effect of lipid association on the local environment of the two tryptophan residues of Locusta migratoria apolipophorin III (apoLp-III) has been studied. In the lipid-free state, Trp115 in helix 4 is buried in the hydrophobic interior of the helix bundle, while Trp130 is located in a loop connecting helices 4 and 5. Fluorescence spectroscopy of single Trp mutants revealed an emission maximum (lambda(max)) of 321 nm for apoLp-III-W@115 (excitation 280 nm) which red-shifted to 327 nm upon binding to dimyristoylphosphatidylcholine (DMPC). ApoLp-III-W@130 displayed a lambda(max) of 338 nm while interaction with DMPC resulted in a blue shift to 331 nm. Quenching studies with KI and acrylamide revealed decreased accessibility to Trp115 compared to Trp130, while lipid binding induced a decrease in quenching of Trp130. Aromatic circular dichroism (CD) spectra showed that Trp vibronic transitions at 278, 286, and 294 nm for lipid-free apoLp-III were caused by Trp115. Upon lipid association, aromatic extrema are reversed in sign, becoming entirely negative with both Trp residues contributing to the vibronic transitions, implying restriction in side-chain mobility of these residues. Thus, lambda(max), quencher accessibility, and aromatic CD analysis indicate that Trp115 is much less solvent-exposed than Trp130. Differences in fluorescence properties of these residues are minimized in the lipid-bound state, a result of relocation of Trp115 and Trp130 into the lipid milieu. Thus, in addition to the hydrophobic faces of apoLp-III amphipathic alpha-helices, the loop region containing Trp130 comes in close contact with DMPC.
authors.affiliation: Lipid Biology Research Group and Protein Engineering Network of Centres of Excellence, Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2S2 Canada.
authors.forename: P M
authors.initials: PM
authors.lastname: Weers
authors.forename: E J
authors.initials: EJ
authors.lastname: Prenner
authors.forename: C
authors.initials: C
authors.lastname: Kay
authors.forename: R O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D020106:Acrylamide / Q000494:pharmacology; D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine; D006110:Grasshoppers; D019791:Guanidine / Q000494:pharmacology; D019476:Insect Proteins / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D009154:Mutation; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D001053:Apolipoproteins; D019476:Insect Proteins; D008055:Lipids; C042470:apolipophorin III; D020106:Acrylamide; D014364:Tryptophan; D014357:Trypsin; D019791:Guanidine; D004134:Dimyristoylphosphatidylcholine