RESUMEN
Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Activación Enzimática/fisiología , Células HeLa , Humanos , Unión Proteica , Dominios Proteicos/fisiología , Multimerización de Proteína , Transporte de Proteínas/fisiologíaRESUMEN
Membrane remodeling is controlled by proteins that can promote the formation of highly curved spherical or cylindrical membranes. How a protein induces these different types of membrane curvature and how cells regulate this process is still unclear. Endophilin A1 is a protein involved in generating endocytotic necks and vesicles during synaptic endocytosis and can transform large vesicles into lipid tubes or small and highly curved vesicles in vitro. By using EM and electron paramagnetic resonance of endophilin A1, we find that tubes are formed by a close interaction with endophilin A1's BIN/amphiphysin/Rvs (BAR) domain and deep insertion of its amphipathic helices. In contrast, vesicles are predominantly stabilized by the shallow insertion of the amphipathic helical wedges with the BAR domain removed from the membrane. By showing that the mechanism of membrane curvature induction is different for vesiculation and tubulation, these data also explain why previous studies arrived at different conclusions with respect to the importance of scaffolding and wedging in the membrane curvature generation of BAR proteins. The Parkinson disease-associated kinase LRRK2 phosphorylates S75 of endophilin A1, a position located in the acyl chain region on tubes and the aqueous environment on vesicles. We find that the phosphomimetic mutation S75D favors vesicle formation by inhibiting this conformational switch, acting to regulate endophilin A1-mediated curvature. As endophilin A1 is part of a protein superfamily, we expect these mechanisms and their regulation by posttranslational modifications to be a general means for controlling different types of membrane curvature in a wide range of processes in vivo.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/metabolismo , Aciltransferasas/genética , Animales , Cristalografía por Rayos X , Dimerización , Electrones , Humanos , Lípidos/química , Liposomas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosforilación/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Marcadores de Spin , Relación Estructura-ActividadRESUMEN
α-Synuclein (αS) is a membrane-binding protein with sequence similarity to apolipoproteins and other lipid-carrying proteins, which are capable of forming lipid-containing nanoparticles, sometimes referred to as "discs." Previously, it has been unclear whether αS also possesses this property. Using cryo-electron microscopy and light scattering, we found that αS can remodel phosphatidylglycerol vesicles into nanoparticles whose shape (ellipsoidal) and dimensions (in the 7-10-nm range) resemble those formed by apolipoproteins. The molar ratio of αS to lipid in nanoparticles is â¼1:20, and αS is oligomeric (including trimers and tetramers). Similar nanoparticles form when αS is added to vesicles of mitochondrial lipids. This observation suggests a mechanism for the previously reported disruption of mitochondrial membranes by αS. Circular dichroism and four-pulse double electron electron resonance experiments revealed that in nanoparticles αS assumes a broken helical conformation distinct from the extended helical conformation adopted when αS is bound to intact vesicles or membrane tubules. We also observed αS-dependent tubule and nanoparticle formation in the presence of oleic acid, implying that αS can interact with fatty acids and lipids in a similar manner. αS-related nanoparticles might play a role in lipid and fatty acid transport functions previously attributed to this protein.
Asunto(s)
Lipoproteínas/química , Nanopartículas/química , alfa-Sinucleína/química , Colesterol/química , Cromatografía en Gel , Microscopía por Crioelectrón , Transferencia Resonante de Energía de Fluorescencia , Humanos , Lipoproteínas/aislamiento & purificación , Lipoproteínas/ultraestructura , Membranas Artificiales , Membranas Mitocondriales/química , Nanopartículas/análisis , Nanopartículas/ultraestructura , Tamaño de la Partícula , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Fosfatidilserinas/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , alfa-Sinucleína/aislamiento & purificación , alfa-Sinucleína/ultraestructuraRESUMEN
α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.
Asunto(s)
Membrana Dobles de Lípidos/química , Micelas , Fosfatidilgliceroles/química , Pliegue de Proteína , alfa-Sinucleína/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Membrana Dobles de Lípidos/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosfatidilgliceroles/metabolismo , Estructura Secundaria de Proteína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two ß-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing ß-sheets that twist around each other. The two ß-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (â¼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.
Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/química , Multimerización de Proteína , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Microscopía Electrónica , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Secundaria de Proteína , alfa-Sinucleína/metabolismoRESUMEN
We characterized the structure of partially unfolded bacteriorhodopsin in sodium dodecyl sulfate (SDS) micelles and compared it with its in vitro refolded structure after reconstitution with dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (DMPC/CHAPS). Intrahelical and interhelical distances were mapped in the protein using strategically located spin-label pairs at helical ends, assayed by pulsed electron paramagnetic resonance spectroscopy (double electron-electron spin resonance, DEER). We find that in SDS the intrahelical end-to-end distances exhibit broad distributions, suggesting a heterogeneous ensemble of conformations with differing secondary structures. Nevertheless, a majority of the denatured population retains end-to-end distances similar to those in the native state. In contrast, the observed greatly increased interhelical distances, in addition to their very broad distributions, suggest that in the SDS micelles very little of the native tertiary structure remains.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Micelas , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Dodecil Sulfato de Sodio/química , Dodecil Sulfato de Sodio/farmacología , Detección de SpinRESUMEN
Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63-86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63-75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic alpha-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.
Asunto(s)
Aciltransferasas/química , Membrana Celular/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Simulación por Computador , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Liposomas/química , Liposomas/metabolismo , Fluidez de la Membrana , Microscopía Electrónica , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Multimerización de Proteína , Ratas , Marcadores de SpinRESUMEN
alpha-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, alpha-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 alpha-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 alpha-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that alpha-synuclein forms an extended, curved alpha-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 alpha-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 A). The alpha-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 alpha-synuclein in the presence of the commonly used membrane-mimetic 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.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Membrana Dobles de Lípidos/química , alfa-Sinucleína/química , Secuencia de Aminoácidos , Simulación por Computador , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Marcadores de SpinRESUMEN
Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein alpha-synuclein (alphaS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS). By combining 18 EPR-derived interelectron spin label distance distributions with NMR-based secondary structure definitions and bond vector restraints, interelectron distances were correlated and a set of theoretical ensemble basis populations was calculated. A minimal set of basis structures, representing the partially folded state of SLAS-bound alphaS, was subsequently derived by back-calculating correlated distance distributions. A surprising variety of well-defined protein-micelle interactions was thus revealed in which the micelle is engulfed by two differently arranged antiparallel alphaS helices. The methodology further provided the population ratios between dominant ensemble structural states, whereas limitation in obtainable structural resolution arose from spin label flexibility and residual uncertainties in secondary structure definitions. To advance the understanding of protein-micelle interactions, the present study concludes by showing that, in marked contrast to secondary structure stability, helix dynamics of SLAS-bound alphaS correlate with the degree of protein-induced departures from free micelle dimensions.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Pliegue de Proteína , alfa-Sinucleína/química , Humanos , Micelas , Modelos Moleculares , Estructura Terciaria de Proteína , Sarcosina/químicaRESUMEN
Amelogenin is a proline-rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self-assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary-secondary structure and self-assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full-length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered beta-structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self-assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (DeltaH(A)) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions.
Asunto(s)
Amelogenina/química , Calorimetría/métodos , Dicroismo Circular/métodos , Temperatura , Amelogenina/genética , Amelogenina/metabolismo , Animales , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.
Asunto(s)
Membrana Celular/química , Modelos Moleculares , Proteínas/química , Secuencia de Aminoácidos , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Unión a Ácidos Grasos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/química , Proteínas de la Membrana , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
The dynamin-related Eps15-homology domain-containing protein 2 (EHD2) is a membrane-remodeling ATPase that regulates the dynamics of caveolae. Here, we established an electron paramagnetic resonance (EPR) approach to characterize structural features of membrane-bound EHD2. We show that residues at the tip of the helical domain can insert into the membrane and may create membrane curvature by a wedging mechanism. Using EPR and X-ray crystallography, we found that the N terminus is folded into a hydrophobic pocket of the GTPase domain in solution and can be released into the membrane. Cryoelectron microscopy demonstrated that the N terminus is not essential for oligomerization of EHD2 into a membrane-anchored scaffold. Instead, we found a function of the N terminus in regulating targeting and stable association of EHD2 to caveolae. Our data uncover an unexpected, membrane-induced regulatory switch in EHD2 and demonstrate the versatility of EPR to study structure and function of dynamin superfamily proteins.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caveolas/metabolismo , Células 3T3-L1 , Animales , Sitios de Unión , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Modelos Moleculares , Pliegue de Proteína , Estabilidad Proteica , Estructura Terciaria de ProteínaRESUMEN
Electron paramagnetic resonance using site-directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r(2) = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.
Asunto(s)
Simulación por Computador , Óxidos de Nitrógeno/química , Proteínas/química , Marcadores de Spin , Aciltransferasas/química , Algoritmos , Animales , Anexinas/química , Drosophila melanogaster , Proteínas de Unión a Ácidos Grasos , Humanos , Proteínas de la Membrana , Modelos Biológicos , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/química , Procesamiento Proteico-Postraduccional , alfa-Sinucleína/químicaRESUMEN
Annexins are soluble proteins that can interact with membranes in a Ca2+-dependent manner. Recent studies have shown that they can also undergo Ca2+-independent membrane interactions that are modulated by pH and phospholipid composition. Here, we investigated the structural changes that occurred during Ca2+-independent interaction of annexin B12 with phospholipid vesicles as a function of pH. Electron paramagnetic resonance analysis of a helical hairpin encompassing the D and E helices in the second repeat of the protein showed that this region refolded and formed a continuous amphipathic alpha helix following Ca2+-independent binding to membranes at mildly acidic pH. At pH 4.0, this helix assumed a transmembrane topography, but at pH approximately 5.0-5.5, it was peripheral and approximately parallel to the membrane. The peripheral form was reversibly converted into the transmembrane form by lowering the pH and vice versa. Furthermore, analysis of vesicles incubated with annexin B12 using freeze-fracture electron microscopy methods showed classical intramembrane particles at pH 4.0 but none at pH 5.3. Together, these data raise the possibility that the peripheral-bound form of annexin B12 could act as a kinetic intermediate in the formation of the transmembrane form of the protein.
Asunto(s)
Anexinas/química , Anexinas/metabolismo , Membrana Celular/metabolismo , Anexinas/genética , Calcio/metabolismo , Técnica de Fractura por Congelación , Concentración de Iones de Hidrógeno , Modelos Moleculares , Unión ProteicaRESUMEN
The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.
Asunto(s)
Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , ADN , Conformación de Ácido Nucleico , Translocación Genética , Anticuerpos Monoclonales/química , Secuencia de Bases , Línea Celular Tumoral , Dicroismo Circular , ADN/química , Humanos , Enlace de Hidrógeno , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Conformación Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Recombinación Genética , Programas Informáticos , Sulfitos/farmacologíaRESUMEN
The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.