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

Abstract

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 located in increments of ~ 5 Å apart 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 X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment suggesting spatial proximity (10 Å to 15 Å) from 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 to study protein spatial organization.

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

“…15 Moreover, as PfFtP77C was uniquely found as a 24-meric assembled cage in solution, the protein was used as a standard independent of cation concentration. 32 …”

“…Thus, in the present paper, we engineered the HumAfFt ferritin to siteselectively introduce two overlapping pyrene moieties at the ferritin dimer interface with the aim of exploring archaeal ferritins association/dissociation mechanism by exploiting pyrene sensitive uorescence emission. Pyrene uorescence properties were widely used for many biological and bioimaging investigations [15][16][17][18][19][20][21][22] as the probe's high extinction coefficient allows studies of proteins in solution at physiologically relevant concentrations and its high stability and long uorescent lifetime give it resistance to photodamage and photobleaching.…”

Apolipoprotein E3-mediated cellular uptake of reconstituted high-density lipoprotein bearing core 3, 10, or 17 nm hydrophobic gold nanoparticles

“…25 Protein purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis using a 4%-20% gradient acrylamide gel; protein concentration was determined by the Bio-Rad DC™ assay or by measuring the absorbance at 280 nm using a molar extinction coefficient of 44,460 M −1 cm…”

Structural analysis of peptide fragments following the hydrolysis of bovine serum albumin by trypsin and chymotrypsin

“…By definition, the excimer is stable only in the excited state and can be observed in the emission but not in the absorption spectrum. The usual model for excimer structure is that of a parallel sandwich model with an interplanar distance of about 0.3-0.5 nm (Bains, Kim, Sorin, & Narayanaswami, 2012;Niwayama, Kassar, Zhao, Sutton, & Altenberg, 2011). Fluorescence decay of excimer can be described as a sum of multi-exponential decays, which is composed of limited number of discrete exponential terms, each of which contains a specific fluorescence lifetime (τ) and a pre-exponential factor as given below (James, Siemiarczuk, & Ware, 1992;James & Ware, 1986;Lakowicz, 2006;Panda & Bhattacharyya, 1992;Pekcan, 1996;Sharma & Schulman, 1999;Siemiarczuk et al, 1990;Stubbs & Williams, 2002;Tkachenko, 2006).…”

“…With 5 heavy atoms in Lys, and 7 heavy atoms in Arg, (only the heavy atoms which are carbon, nitrogen, and oxygen, were counted), there exist 35 (= 5 × 7) possible pairwise distances to be evaluated. The Arg-Lys pair was then considered as a potential site for an excimer, if it holds at least 4 pairwise distances that are less than 5 Å, which is usually selected as the most favorable distance for observing an excimer in some experimental studies (Bains et al, 2012;Niwayama et al, 2011). After treating all 3403 residue pairs which accounts to 103,579 distance evaluations, only 9 residue (or PM) pairs were found to be in a favorable orientation for generating a potential excimer.…”