Robert M. Henderson

Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize

P2X receptors are cation-selective channels activated by extracellular ATP. The architecture of these receptors is still not completely clear. Here we have addressed this issue by both chemical cross-linking and direct imaging of individual receptors by atomic force microscopy (AFM). Cross-linking of the P2X2 receptor produced higher order adducts, consistent with the presence of trimers. The mean molecular volume of the receptor determined by AFM (409 nm3) also points to a trimeric structure. P2X2 receptors bearing His6 epitope tags were incubated with anti-His6 antibodies, and the resultant complexes were imaged by AFM. For receptors with two bound antibodies, the mean angle between the antibodies was 123°, again indicating that the receptor is a trimer. In contrast, cross-linking of the P2X6 receptor did not produce higher order adducts, and the mean molecular volume of the receptor was 145 nm3. We conclude that P2X2 receptors are trimers, whereas the P2X6 receptor subunits do not form stable oligomers.

Real-Time Analysis of the Effects of Cholesterol on Lipid Raft Behavior Using Atomic Force Microscopy

Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping

Many DNA-modifying enzymes act in a manner that requires communication between two noncontiguous DNA sites. These sites can be brought into contact either by a diffusion-mediated chance interaction between enzymes bound at the two sites, or by active translocation of the intervening DNA by a site-bound enzyme. EcoP15I, a type III restriction enzyme, needs to interact with two recognition sites separated by up to 3,500 bp before it can cleave DNA. Here, we have studied the behavior of EcoP15I, using a novel fast-scan atomic force microscope, which uses a miniaturized cantilever and scan stage to reduce the mechanical response time of the cantilever and to prevent the onset of resonant motion at high scan speeds. With this instrument, we were able to achieve scan rates of up to 10 frames per s under fluid. The improved time resolution allowed us to image EcoP15I in real time at scan rates of 1–3 frames per s. EcoP15I translocated DNA in an ATP-dependent manner, at a rate of 79 ± 33 bp/s. The accumulation of supercoiling, as a consequence of movement of EcoP15I along the DNA, could also be observed. EcoP15I bound to its recognition site was also seen to make nonspecific contacts with other DNA sites, thus forming DNA loops and reducing the distance between the two recognition sites. On the basis of our results, we conclude that EcoP15I uses two distinct mechanisms to communicate between two recognition sites: diffusive DNA loop formation and ATPase-driven translocation of the intervening DNA contour.

Direct observation of DNA translocation and cleavage by the EcoKI endonuclease using atomic force microscopy

Direct observation of DNA translocation and cleavage by the Eco KI endonuclease using atomic force microscopy

Atomic Force Microscopy Reveals the Stoichiometry and Subunit Arrangement of the α4β3δ GABAA Receptor

The GABAA receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains α1-, β2-, and γ2-subunits in the arrangement αβαγβ, counter-clockwise around the pore. GABAA receptors containing δ-instead of γ-subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the α4β3δ form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2α:2β:1δ. The distributions of angles between pairs of antibodies against the α- and β-subunits both had peaks at around 144°, indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the β-subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the δ-subunit and Fab fragments against the α-subunits indicates a predominant subunit arrangement of αβαδβ, counter-clockwise around the pore when viewed from the extracellular space.

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 +/- 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His(6)-tagged sigma-1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131 degrees . When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134 degrees , confirming that the small particles were sigma-1 receptors. Of interest, we found that the sigma-1 receptor ligand haloperidol caused an approximately 50% reduction in ASIC1a/sigma-receptor binding, suggesting a way in which sigma-1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the sigma-1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 sigma-1 receptor/1 ASIC1a subunit.

Direct visualization of G-quadruplexes in DNA using atomic force microscopy

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sγ3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.

Direct visualization of ligand-protein interactions using atomic force microscopy.

Streptavidin is a 60‐kDa tetramer which binds four molecules of biotin with extremely high affinity (KA∼1014 M−1). We have used atomic force microscopy (AFM) to visualize this ligand‐protein interaction directly. Biotin was tagged with a short (152‐basepair; 50‐nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105±3 nm3) was in close agreement with the value calculated from its molecular mass (114 nm3). Biotinylation increased the apparent size of streptavidin (to 133±2 nm3), concomitant with an increase in the thermal stability of the tetramer. Images of streptavidin with one to four molecules of DNA‐biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA‐biotin. The slight under‐representation of higher occupancy states supported this idea. Streptavidin with a single molecule of DNA‐biotin bound was used to tag biotinylated β‐galactosidase, a model multimeric enzyme. The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA‐biotin/streptavidin tags could potentially shed light on the architecture of multi‐subunit proteins.