Lista de Chequeo

While the probing of PSI [Red] was attempted via the removal of methyl viologen, the extent of illumination from the AFM laser meant that the P700 in PSI remained oxidised for significant periods. Although the use of larger cantilevers for the AFM probe may alleviate this issue, effectively shielding the surface underneath the probe, this could lead to either a loss of force resolution or higher noise, both of which are undesirable for these experiments. As such, it was assumed that we could not exert any control over the redox state of PSI on the surface. Future experiments could utilise an AFM laser at a longer wavelength where PSI doesn’t absorb to understand the effect of light on the interaction.

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The effect of the redox state of Pc however could be probed in our experiments, by incubation with reducing or oxidising agents prior to imaging. In addition, the exclusion of MV was made to ensure no electrons from PSI could be accepted by MV and donated to Pc, skewing the redox state being probed. The 2-fold change observed in the Pc[Ox] state relative to the Pc[Red] state is in agreement with the previous experiments performed to determine differences in the kon (Drepper et al., 1996). This same

study also found a 4.5-fold difference in the koff, with the Pc[Ox] form dissociating faster than the

Pc[Red] form, suggesting a redox selectivity in the system. The unbinding force can be correlated to the dissociation rate constant for the interaction (section 1.4.2), and as such a difference would be expected in the unbinding forces. The extent of this however would depend on the change in the reaction coordinate between these states (section 1.4.2). No change was observed in our experiments in the distribution of the unbinding forces measured, and again this may simply reflect the poor signal to noise ratio in the experiments obscuring a change below the threshold. Alternatively, the manifestation of the change in the unbinding forces may have been too small to show effect here, as a number of parameters in the Bell-Evans model for unbinding force are not known for PSI : Pc. While this work explored a number of features for the PSI : Pc binding, it faced limitations from the low unbinding force of the interaction. While lower noise cantilevers for AFM probes could potentially be used, the high expense of the PeakForce-HIRS-SSB probes used in these experiments already was a limiting factor. A more prudent avenue forward would be the assessment of mutants of PSI previously characterised by bulk phase measurements, such as the mutants in Kuhlgert et al., 2012 which saw large increases in affinity from mutation of charged residues surrounding the hydrophobic binding interface.

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