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Kinetics of small molecule interactions with membrane proteins in single cells measured with mechanical amplification

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Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult

Measuring small molecule interactions with membrane proteins in single cells is critical for understanding many cellular processes and for screening drugs. However, developing such a capability has been a difficult challenge. We show that molecular interactions with membrane proteins induce a mechanical deformation in the cellular membrane, and real-time monitoring of the deformation with subnanometer resolution allows quantitative analysis of small molecule–membrane protein interaction kinetics in single cells. This new strategy provides mechanical amplification of small binding signals, making it possible to detect small molecule interactions with membrane proteins. This capability, together with spatial resolution, also allows the study of the heterogeneous nature of cells by analyzing the interaction kinetics variability between different cells and between different regions of a single cell.

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  • 2015-10-23

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Methods for detection of small molecule-protein interactions

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Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the

Detection of molecular interactions is critical for understanding many biological processes, for detecting disease biomarkers, and for screening drug candidates. Fluorescence-based approach can be problematic, especially when applied to the detection of small molecules. Various label-free techniques, such as surface plasmon resonance technique are sensitive to mass, making it extremely challenging to detect small molecules. In this thesis, novel detection methods for molecular interactions are described.

First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.

Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.

Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.

Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.

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  • 2015