Experimental study on molecular fluctuations of biomolecules by force spectroscopy

Atomic force microscopy (AFM) measurements of biomolecules, such as proteins, are often affected by molecular fluctuations. Not only due to the intrinsic thermal fluctuation, deformation and displacement of a part or the whole molecule are induced by the AFM tip, especially when the molecules are weakly fixed to a substrate. Here we have studied the effect of the molecular fluctuations on the topography of the streptavidin (SA) protein molecules bound to a DNA origami in two different configurations by force spectroscopy experiments. While the experimental and theoretical height histograms of a tightly-fixed SA molecule were in good agreement, the experimental height histogram of a weakly-fixed SA molecule was distorted. We discussed the origins of the discrepancy of the histogram for the latter and suggested that it was due to the tip-induced deformation or the displacement of the molecule. The methodology presented here is useful for assessment of the appropriate fixation conditions for high-resolution topographic and functional imaging experiments of a single molecule by AFM.