Single Molecule Measurements of the Accessibility of Molecular Surfaces

Abstract

An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method (“Q-SLIP”, Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.

A Toxicogenic Interaction between Intracellular Amyloid-β and Apolipoprotein-E

“…One of the most powerful methods to probe the difference in the lifetime of the species in combination with their diffusion times is the recently developed technique of two-dimensional fluorescence lifetime correlation spectroscopy or 2DFLCS . 2D FLCS can distinguish different species in a mixture based on their lifetimes and sizes, and reports their interconversion dynamics with submicrosecond time resolution. − Furthermore, subsequent filtered FCS analysis can provide species-associated correlation curves, hence the size (diffusion time) of each species. , Additionally, the stoichiometry of the oligomers can be tested using the single molecule photobleaching (smPB) technique. , …”

“…32,34 Additionally, the stoichiometry of the oligomers can be tested using the single molecule photobleaching (smPB) technique. 35,36 To facilitate such detailed characterization, extracts from cells pretreated with RAβ were subjected to 2D FLCS and smPB measurements. Extraction of the oligomers did not cause major perturbation of their intracellular state, as was confirmed by lifetime measurements (Figure 2A (MEM) 37 based lifetime distribution of RAβ oligomers (prepared freshly in buffer vs extracted from different cells) are shown in Figure 2B.…”

Single-molecule Mapping of Amyloid-β Oligomer Insertion into Lipid Bilayers

“…Second, a lack of proper experimental strategy to correlate the membrane interaction with the toxicity of each type of oligomer. Here we addressed the first problem with a newly devised single-molecule technique called Q-SLIP, which allows the simultaneous determination of the stoichiometry of each individual oligomer in a mixture, and the surface exposure of each individual monomer in each type of oligomer 34 ( vide infra ). Next to address the second problem, we adopted an indirect approach.…”

“…However, smPB alone cannot provide any information about conformation or membrane penetration of the oligomers. This problem can be tackled by the recently developed method of Quencher Induced Step Length Increase in Photobleaching (QSLIP) 34 . Processes such as dynamic quenching and labile complexation with a quencher can impart photostability to a fluorophore, since quenching reduces its probability to be in the singlet or triplet excited state from where photobleaching typically occurs 41 .…”

Single-Molecule Maps of Membrane Insertion by Amyloid-β Oligomers Predict Their Toxicity

“…Here we addressed the first part with the single-molecule technique called Quencherinduced step length increase in photobleaching (QSLIP) recently demonstrated by us. 42 This allows the simultaneous determination of the stoichiometry of each individual oligomer in a mixture, and also the surface exposure of a fluorophore attached to a specific part (e.g., the N-terminus) of each monomer in a given type of oligomer using contact-based fluorescence quenchers (vide infra). Quenching can impart photostability to a fluorophore by reducing its probability of being in the excited state, especially in the triplet excited state (from where photobleaching typically occurs).…”