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Molecular mechanisms and interactions

Challenge

Molecular interactions means the dynamic interaction between two or more labeled molecules. In cells, advanced microscopy images are used to understand the relationship between molecules through their spatial organization. For example, interest is growing in the underlying mechanisms of membrane contact sites (MCS) in cellular organelles. However, often the most important aspect of an interaction is the rate of binding, the dissociation constant, the dynamics and dwell times. This quantitative information is especially useful for understanding the effect of inhibitors and small molecules or solution conditions on the behavior of a particular interaction, such as the activity of an enzyme induced by substrate binding.

Solution with nanoimager

The Nanoimager can determine the dynamics of single molecular interactions in real time. By measuring single molecules, it avoids the inherent averaging of other ensemble techniques such as ensemble FRET, calorimetry or fluorimetry. Therefore, any sub-populations of molecules and any dynamic intermediates, which are often found in molecular interactions, can be detected with the Nanoimager.

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Case study

One approach used by the Nanoimager for extracting this information is single-molecule FRET. smFRET is a technique that involves labeling the sample with two fluorophores, which can be on the same or different molecular species, to measure a dynamic process (intra- and inter-molecular processes respectively). FRET acts as a real-time dynamic ruler on the 2-10 nm range, so it can detect two proteins binding and unbinding for example. It can also detect the conformational changes in a single enzyme, and how these transitions are affected by binding substrates.

The Nanoimager can measure these single-molecule interactions, and quantitate them, for various types of sample: molecules bound to a surface, in solution, on an artificial membrane or on a cell membrane close to a glass coverslip. Using TIRF, smFRET can be performed on live cell membranes with the Nanoimager.

In other methods, the Nanoimager can characterize molecular interactions using dynamic colocalization of two labeled species, monitoring their position over time.