␣-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, ␣-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of ␣-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of ␣-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that ␣-synuclein forms an extended, curved ␣-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like ␣-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd ‫؍‬ 0.82 Å). The ␣-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of ␣-synuclein in the presence of the commonly used membranemimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment.EPR ͉ Parkinson's disease ͉ fibril-forming proteins ͉ 11-aa repeats T he interaction of ␣-synuclein with membranes is thought to be important in its physiologic function in vivo, as well as in its misfolding and aggregation in the pathogenesis of Parkinson disease (1-10). Although the function of ␣-synuclein in vivo is not fully understood, it has been observed to localize to presynaptic nerve termini, where it modulates presynaptic pool size and neurotransmitter release (11-16). These functions are likely to be mediated by the interaction of ␣-synuclein with synaptic vesicles, and in vitro studies have shown that ␣-synuclein interacts strongly with highly curved vesicles that are similar in size to synaptic vesicles (17, 18). The structural characterization of membrane-bound ␣-synuclein is significant, given the importance of membrane interactions to the pathologic and physiologic roles of ␣-synuclein.Previous studies have revealed that the interaction of monomeric ␣-synuclein with negatively charged vesicles induces a predominantly ␣-helical structure located in the N-terminal region of the protein (17,19,20). This region contains seven 11-aa-repeat regions that share some sequence similarity with apolipoproteins [supporting information (SI) Fig. S1]. Sequence analysis using algorithms for apolipoproteins predicts the formation of five separate helices (17). However, no high-resolutio...

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In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 Å in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 Å, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA–protein complexes.

Background: Human islet amyloid polypeptide (hIAPP) fibrils of unknown structure are formed in type 2 diabetes. Results: A hIAPP fibril structure was derived from EPR data, electron microscopy, and computer modeling. Conclusion:The fibril is a left-handed helix that contains hIAPP monomers in a staggered conformation. Significance: The results provide the basis for therapeutic prevention of fibril formation and growth.

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron-electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.