AbstractThe type I R-M enzyme EcoR124I is a multifunctional, multisubunit molecular motor with the ability to self-assemble. In the presence of hydrophobic compounds, subunit disassembly has been observed leading to the possibility of using the enzyme as a nanoactuator in toxicity biosensors.
A better understanding of single molecule interactions between the subunits has been investigated using atomic force microscopy (AFM), a powerful tool for measuring forces and dynamics between single molecules with a picoNewton sensitivity.
AFM imaging of DNA fragments with a single recognition binding site for EcoR124I positioned in the middle or at 1/3 of the length of DNA, was used to study the assembled holoenzyme. Reproducible DNA imaging was investigated using divalent cations (Mg2+, Ca2+, Ni2+). The presence of only one EcoR124I holoenzyme bound to DNA was observed, confirming the specificity of binding. Molecular volume (Vm) measurements were used to identify subunits and complexes. The effect of ATP analogues (ATP-γ-S and AMP-pnp) on enzyme stability was also investigated. The addition of ATP, although not novel, confirmed the enzyme activity by showing the ability of the enzyme to translocate.
Biotin-avidin interactions were studied using AFM force curves as a model to probe the novel HsdR-MTase system. AFM tips were functionalised using both glutaraldehyde and a PEG linker. In the former, many multiple event force curves were seen, although the final “pull-off” event yielded information on single-molecule or near single-molecule interactions: a single biotin-avidin interaction at 56 ± 13 pN was measured, with further periodic force maxima at 98 ± 15 and 161 ± 3 pN (two and three interactions, respectively). The use of a PEG linker allowed more sensitive measurements to be made, with a single biotin-avidin interaction at 47 ± 9.5 pN and, again, periodic maxima were seen at 93 ± 7 and 143 ± 4 pN. The PEG linker method allowed more single molecules interactions to be measured (ca. 70% of analysed forcedistance curves).
Forces between a GST-HsdR(PrrI) motor subunit attached to an AFM tip using a PEG linker and MTase on poly-L-lysine pre-treated mica were studied using dynamic force spectroscopy (DFS). A single barrier in the energy landscape of the complex was found in the dissociation pathway (xdiss) to be located 13.5 Å from the bound state. The value kdiss for the GST-HsdR(PrrI)-MTase complex was calculated to be 0.16 s-1 and the lifetime Ʈ(0) of the GST-HsdR(PrrI)-MTase bond was found to be 6.25 s.
GST – anti GST antibody interactions and HsdR – anti-GST antibody interactions suggest that forces measured between HsdR and MTase were realistic for the GSTHsdR(PrrI)-MTase complex.
|Date of Award||Nov 2010|
|Supervisor||Keith Firman (Supervisor), James Smith (Supervisor) & Sheelagh A. Campbell (Supervisor)|