Solvent Accessibility Of Individual Protein Molecules And Molecular Complexes Measured At A Single Molecule Level

Solvent accessibility of different parts of a protein or a molecular complex is an important measure of its conformation and/or membrane insertion. While there are standard techniques available for bulk measurements, it is hard to do for single molecules. Here, by combining single molecule photobleaching (smPB) and fluorescence quenching techniques, we demonstrate a method that can measure the solvent exposure of membrane-attached proteins at a single molecule level. In smPB, an individual molecule or an oligomeric assembly can be characterized by counting the number of bleaching steps1,2. But, additional information is available in terms of the bleaching step length (BSL), i.e. how long the molecule stays unbleached. If we have a small molecule quencher that can effectively quench the excited singlet or the triplet state, either by collision or by ground state complex formation (or both), the photostability and hence the BSL of the fluorophore should increase. Thus the accessibility of the chromophore to the quencher can be measured by measuring the change in the BSL. We observed that a rhodamine-labeled lipid and a rhodamine-labeled lipid-tailed peptide showed a near two-fold increase in the BSL upon addition of tryptophan (quencher) in aqueous solution. We then applied this to measure the solvent exposure of membrane-bound monomers of amylin (hIAPP), which are rhodamine-labeled at the N-terminal. It showed only a 20%increase in BSL, indicating a lower level of solvent exposure of the N-terminal. This demonstrates that we can measure the difference in the level of tryptophan-accessible exposure of the same dye label in different molecular environments. 1) Dey et al. Biophysical Journal 2020,118, 1101-1108 2) Dey et al. Phys. Chem. Chem. Phys., 2020, 22, 14613-14620