Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping

Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor

The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(?35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation

Carbon nanotube atomic force microscopy

This thesis concerns the manufacture of carbon nanotube atomic force microscope (NTAFM) probes and their employment in the high-resolution imaging of biological macromolecules. Attention was focused initially on synthesis of carbon nanotubes and the refinement of the growth processes to obtain nanotubes of controlled dimensions. These growth processes were subsequently used to grow nanotubes directly onto AFM tips, followed by attempts at controlling the dimensions of these directly-grown nanotubes. Individually fabricated NTAFM probes are also described, along with attempts to optimise the strength of the AFM probe-nanotube interaction through the use of various hydrophobic coatings. NTAFM probes produced by both techniques, but predominantly through individually assembled probes using hydrophobic coatings, were used to image a range of natural and synthetic nucleic acid molecules and investigate the influence of the use of a nanotube probe on the dimensions observed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Harnessing catalysis to enhance scanning probe nanolithography

The use of scanning probes bearing catalysts to perform surface nanolithography combines the exquisite spatial precision of scanning probe microscopy with the synthetic capabilities of (bio)chemical catalysis.</p

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs

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

The σ-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/σ-1 receptor complex. When isolated His8-tagged ASIC1a was imaged in complex with anti-His6 antibodies, the angle between pairs of bound antibodies was 135°, consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His6-tagged σ-1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131°. When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134°, confirming that the small particles were σ-1 receptors. Of interest, we found that the σ-1 receptor ligand haloperidol caused an ∼50% reduction in ASIC1a/σ-receptor binding, suggesting a way in which σ-1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the σ-1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 σ-1 receptor/1 ASIC1a subunit