Pyrene excimer fluorescence: a spatially sensitive probe to monitor lipid-induced helical rearrangement of apolipophorin III. Biochemistry 2000 Jun 06;39(22):6594-601
Date
06/01/2000Pubmed ID
10828977DOI
10.1021/bi992609mScopus ID
2-s2.0-0034612303 (requires institutional sign-in at Scopus site) 122 CitationsAbstract
Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid binding-induced helical rearrangements, we exploited the unique fluorescence characteristics of N-(1-pyrene)maleimide. Pyrene is a spatially sensitive extrinsic fluorescent probe, which forms excited-state dimers (excimers) upon close encounter with another pyrene molecule. Cysteine residues were introduced into apoLp-III (which otherwise lacks cysteine) at Asn 40 (helix 2) and/or Leu 90 (helix 3), creating two single-cysteine mutants (N40C-apoLp-III and L90C-apoLp-III) and N40C/L90C-apoLp-III, a double-cysteine mutant, which were labeled with pyrene maleimide. Pyrene-labeled N40C/L90C-apoLp-III, but not the pyrene-labeled single-cysteine mutants, exhibited strong excimer fluorescence in the lipid-free, monomeric state. Guanidine hydrochloride titration and temperature studies revealed a loss in excimer fluorescence, accompanied by a loss in the molar ellipticity of the protein. When apoLp-III interacts with phospholipid vesicles to form disklike complexes, a significant loss in excimer fluorescence was noted, indicating that the helices bearing the pyrene moieties diverge from each other. Pyrene excimer fluorescence was further employed to examine the relative orientation of lipid-bound apoLp-III molecules. Pyrene-labeled N40C- or L90C-apoLp-III displayed no excimer fluorescence in the disk complexes, while complexes prepared with an equal mixture of both single-labeled mutants did emit excimer fluorescence, indicating apoLp-III adopts a preferred nonrandom orientation around the perimeter of the bilayer disk. These studies establish pyrene excimer fluorescence as a useful spectroscopic tool to address intra- and intermolecular interactions of exchangeable apolipoproteins upon binding to lipid.
Author List
Sahoo D, Narayanaswami V, Kay CM, Ryan ROAuthor
Daisy Sahoo PhD Dean, Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApolipoproteins
Circular Dichroism
Dimerization
Fluorescent Dyes
Guanidine
Insect Proteins
Lipids
Liposomes
Maleimides
Manduca
Mutation
Protein Structure, Secondary
Recombinant Proteins
Spectrometry, Fluorescence
Temperature
Trifluoroethanol