The extent of pyrene excimer fluorescence emission is a reflector of distance and flexibility: analysis of the segment linking the LDL receptor-binding and tetramerization domains of apolipoprotein E3.
Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375-405 nm) and an additional band (called excimer) at ~460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an α-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys residues separated by ~5 Å increments on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were ~5 Å from each other with an e/m ratio of ~3.0, which decreased to ~1.0 at 20 Å. An inverse correlation between e/m ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 Å (4.9 Å from NMR and 5.7 Å from the X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment, suggesting spatial proximity (10-15 Å) to corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe for studying protein spatial organization.
Department of Chemistry and Biochemistry, 1250 Bellflower Boulevard, California State University Long Beach, Long Beach, CA 90840, USA.
Gursharan K
GK
Bains
Sea H
SH
Kim
Eric J
EJ
Sorin
Vasanthy
V
Narayanaswami
D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000737:chemistry*; D011973:Receptors, LDL / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D053318:Apolipoprotein E3; D005456:Fluorescent Dyes; D008301:Maleimides; D011721:Pyrenes; D011973:Receptors, LDL; C043592:maleimide; C030984:pyrene