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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.

SUBMITTER: Dey A

PROVIDER: S-EPMC8672140 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence P(n)CG(3)K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Förster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models-including the worm-like chain, the freely jointed chain, and the self-avoiding chain-could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 Å, ~67 Å, ~51 Å, and ~76 Å for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P(8) exists as the extended all-trans isomaer whereas P(12) and P(15) predominantly contained one proline residue in the cis conformation. P(24) exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules.

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Single Molecule Measurements of the Accessibility of Molecular Surfaces.

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