An EPR "spectroscopic ruler" was developed using a series of a-helical polypeptides, each modified with two nitroxide spin labels. The EPR line broadening due to electron-electron dipolar interactions in the frozen state was determined using the Fourier deconvolution method. These dipolar spectra were then used to estimate the distances between the two nitroxides separated by 8-25 A. Results agreed well with a simple a-helical model. The standard deviation from the model system was 0.9 A in the range of [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] A. This technique is applicable to complex systems such as membrane receptors and channels, which are difficult to access with high-resolution NMR or x-ray crystallography, and is expected to be particularly useful for systems for which optical methods are hampered by the presence of lightinterfering membranes or chromophores.Many studies in structural biology are dependent on the physical techniques to measure distances in proteins and nucleic acids. X-ray crystallography and high-resolution NMR have been useful in determining the three-dimensional structures of relatively simple biological macromolecules. For complex systems such as membrane proteins, fluorescence energy transfer (FET) has been the main alternative for measuring distances up to 80 A. FET has been successful for studies of intermolecular organization in biological systems (1, 2), ligandreceptor interactions (3), and structures of nucleic acids (4).Recently, site-directed spin labeling EPR has become useful for studying proteins (5, 6). One or two native residues are mutated to cysteines, which are then labeled with thiol-specific nitroxide spin labels. This technique can also be used to study local secondary structure (7,8). Nucleic acids also appear to be amenable to spin labeling (9). Although spin labeling has been used to estimate distances in the past (10-12), no EPR "spectroscopic ruler" similar to that developed by Stryer and Haugland (13) for FET has been constructed or tested on model systems.In this work a convenient and accurate EPR method to determine distances between two site-specifically placed nitroxides in the range of 8-25 A in biomacromolecules is presented. In this method the pure dipolar spectrum for two interacting spins in the frozen state is directly Fourier deconvoluted from the dipolar broadened continuous-wave EPR spectrum. The average interspin distance and the variance of its distribution are obtained from this dipolar spectrum.The method was tested using a-helical peptides as a model system. The peptides were alanine-based helices with spinlabeled cysteines substituted for alanines at two locations from 1 to 13 residues apart. There is excellent agreement between the spin-spin distances from a simple model and experimental results in the range of 8-25 A. It is also shown that this method is useful for systems that have impurities of singly labeled species. Although this methodology is complementary to FET, EPR has the advantages of easier...

The heterotrimeric synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of the synaptic vesicle-associated membrane protein 2 (VAMP2) and presynaptic plasma membrane proteins SNAP-25 (synaptosome-associated protein of 25,000 Mr) and syntaxin 1A, is a critical component of the exocytotic machinery. We have used spin labeling electron paramagnetic resonance spectroscopy to investigate the structural organization of this complex, particularly the two predicted helical domains contributed by SNAP-25. Our results indicate that the N- and C-terminal domains of SNAP-25 are parallel to each other and to the C-terminal domain of syntaxin 1A. Based on these findings, we propose a parallel four-stranded coiled coil model for the structure of the synaptic SNARE complex.

Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions. Here we show that fusion driven by neuronal SNAREs is inhibited by the regulatory protein complexin. Furthermore, inner-leaflet mixing is strongly impaired relative to total lipid mixing, indicating that inhibition by complexin arrests fusion at hemifusion. When the calcium sensor synaptotagmin is added in the presence of calcium, inhibition by complexin is relieved and full fusion rapidly proceeds.

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.