Abstract
The flexibility of tethered molecules, such as those bound to biological membranes, is an important property that can influence molecular height, mobility, and accessibility. However, quantifying the flexibility of surface-tethered biomolecules in aqueous environments has been difficult due to a lack of experimental tools. Here, we introduce SurFlex microscopy, a method based on fluorescence anisotropy that exploits the relationship between the conformational dynamics of a tethered molecule and the rotational diffusion of an attached fluorophore to extract information about molecular flexibility. By analyzing the polarization state of photons emitted after polarized excitation, we quantify apparent molecular flexibilities that include effects of tethering, self-interactions, and buffer conditions. We first demonstrate the capabilities of SurFlex microscopy by measuring the flexibility of bilayer-tethered single-stranded DNA (ssDNA) of different lengths and nucleotide sequences. We find that sequence significantly impacts ssDNA flexibility, consistent with theoretical estimates, with weak intramolecular interactions in random sequences leading to higher apparent stiffness. Interestingly, we show that a pathological DNA sequence linked to Huntington's disease exhibits unusual flexibility despite intramolecular interactions. We next extend SurFlex microscopy to live cells by measuring surface glycoprotein flexibility on red blood cells using fluorescent lectins. We show that trypsinization decreases glycan fluctuations, demonstrating that modifications to the cell surface can alter the flexibility of remaining surface molecules. SurFlex microscopy provides a tool for quantifying molecular flexibility that can be used to study the role of tethered surface molecules in fundamental biological processes.