The molecular pathogenesis of disorders arising from protein mis-folding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex mis-folding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease, we have developed a TOAC spin-labeled Aβ peptide to observe its isoform-dependent interaction with the apoE protein. While most individuals carry the E3 isoform of apoE, approximately 15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for Alzheimer's disease. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We employed EPR spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin labeled side chain report on side chain and backbone dynamics, as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ.

Pulsed Electron Double Resonance (PELDOR)/Double Electron-Electron Resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the 4-pulse DEER sequence and can often require up to 12+ hours of signal averaging for biological samples (depending upon spin label concentration). In this rapid report, we present for the first time, a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to Xband. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents 169-fold decrease in data-collection time, reveals greatly improved frequency spectrum, higher quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.Pulsed Electron Double Resonance (PELDOR)/Double Electron-Electron Resonance (DEER) spectroscopy is a rapidly emerging, powerful structural biology technique in which the dipolar coupling between two unpaired electron spins (usually site-directed nitroxide spin labels) is measured(1-3). The strength of the dipolar coupling can then be used to determine the distance between the two spins in the range of 2-8 nm(4-9). This allows researchers to gain valuable structural information from samples in which other techniques like solution NMR or X-ray crystallography prove difficult or impossible(10-12). These measurements are typically conducted with X-band (9.4 GHz) microwave excitation using the 4-pulse DEER sequence and can often require up to 12+ hours of signal averaging (depending upon concentration) for typical biological samples. In this communication, we report for the first time a substantial increase in sensitivity, that is obtained by collecting DEER spectra using a pulse Q-band (34 GHz) EPR spectrometer. The huge boost in sensitivity at Q-band reveals higher quality data and significantly reduces data acquisition time.For this study, an α-helical coiled coil peptide with a Leucine Zipper (LZ) motif (residues 245-281) of the yeast transcriptional activator GCN4(13,14) (PDB entry 1YSA) was used as a model. The peptide was synthesized on a solid-state peptide synthesizer with a single TOAC nitroxide spin label at position 248 with Gln→TOAC substitution for the distance measurements between the two monomers. The TOAC(15-19) spin label was chosen over the traditional MTSL (1,14) to eliminate disproportionation of the two label disulfide bonds to * To whom correspondence is to be addressed: Gary A. Lorigan Email: garylorigan@muohio.edu, Tel: 513-529-3338, Fax: 513-529-5715. form a linked peptide dimer, and to avoid the motional flexibility of the MTSL nitroxide group for more accurate inter-coil backbone distance measurements. In previously published r...

BackgroundThe deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.Methodology/Principal FindingsTo achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.ConclusionsFluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.

A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 14.6 Å from a dual TOAC-labeled AChR M2δ peptide at positions 7 and 13 that closely matches with the 14.5 Å distance obtained from a model of the labeled AChR M2δ peptide. In addition, the angles determining the relative orientation of the two nitroxides have been determined, and the results compare favorably with molecular modeling. The global analysis of the data from the aligned samples gives much more precise estimates of the parameters defining the geometry of the two labels than can be obtained from a randomly dispersed sample.

Wild-type Phospholamban (WT-PLB), a Ca2+-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca2+-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13° ± 4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100 ns rotational correlation time (τc); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.