Abstract
We have recently demonstrated measurements of relative distances between individual, spectrally distinct fluorescent probes that are attached on a biological molecule with precision and accuracy <1nm. Here we extend this technique to measure relative distances between two (or more) fluorophores of the same spectral characteristics.We show that the improved resolution afforded by our microscope imaging system allows characterizing biological structures at the molecular scale. In one application we applied our technique to image individual dimers of Endothelial cadherin (E-cadherin) molecules. At 1mM Ca++ we find the majority of the dimers in an extended configuration, consistent with the crystal structure of the full C-cadherin ectodomain. Interestingly, we observe a separate population in a less extended configuration, possibly related to flexibility of the E-cadherin binding interface. As a function of the free Ca++ concentration we observe a continuous, cooperative transition of the extension of the molecules from the rigid extended configuration to a collapsed state. The apparent Kd=70uM and degree of cooperativity ∼1.5 from our measurements are consistent with various previous indirect investigations (e.g. intrinsic fluorescence, proteolytic sensitivity, circular dichroism, NMR etc).These results clearly demonstrate the power of our approach to image individual molecular scale structures and detect conformational changes at the nanometer scale, establishing it as a unique Structural Biology tool, that can operating in ambient, physiological conditions and is based exclusively on optical far-field fluorescence imaging.
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