Label-Free Single-Molecule Conalbumin Analysis

Nanoaperture optical tweezers (NOTs) were used to analyze conalbumin in various forms. By analyzing the power spectrum of the NOT-transmitted laser signal, differences between iron and iron-free conalbumin were observed; the corner frequency extrapolated to zero-laser power was significantly larger in magnitude for conalbumin with iron, which was interpreted as coming from the enhanced electrostatic interactions close to the surface of the nanoaperture. Conalbumin in a diluted, but otherwise unprocessed, egg white sample showed the same behavior as purified iron-free conalbumin. Dynamic two-state transitions in the NOT signal were observed for iron-free conalbumin and conalbumin in egg white samples. We used this to determine the dominant state as a function of temperature, with one state showing a maximum occupancy around 30.4 °C. Deconvolution of the probability distribution function was used to find the energy landscape associated with this two-state transition. This work shows the potential of NOTs to see variations with metal ion binding, including conformational dynamics related to the binding at timescales not accessible to other methods.