Fluorescence studies of lipid association-induced conformational adaptations of an exchangeable amphipathic apolipoprotein.
The conformational adaptability of Manduca sexta apolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein. M. sexta apoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145 is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145 and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKa of Tyr145 in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the (lambda)max of absorbance of Tyr145 was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145 fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145 in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145 exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145 remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145 which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145 is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145 distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.
Narayanaswami
V
V
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Frolov
A
A
Schroeder
F
F
Oikawa
K
K
Kay
C M
CM
Ryan
R O
RO
D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D018402:Manduca; D015394:Molecular Structure; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000737:chemistry
D001053:Apolipoproteins; D002352:Carrier Proteins; D008055:Lipids; C042470:apolipophorin III; D014443:Tyrosine