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Real-Time Angstrom-Resolution Imaging of Proteins Using Light Microscopy

Description:

A strategically-designed light microscope setup with data analysis software resolves angstrom-scale protein conformational changes in real time.

 

Inventor

Zhe Lu and John H. Lewis

 

Problem

Protein conformational changes are intricately involved in their mechanisms of action. This information is key to development of new therapeutic strategies to treat diseases. Current methods, however, are limited in their abilities to resolve protein movements in real time. X-ray crystallography and cryo-electron microscopy provide angstrom-resolution, but lack meaningful time resolution. Current light microscopy techniques may be time resolved, but lack sufficient spatial resolution to resolve small conformational changes.

 

Solution

This technology describes the successful application of a visible-light microscopic technique to resolve angstrom-scale protein conformational changes in real time. The inventors have demonstrated that the orientation of an element of secondary structure may be tracked by attaching a fluorophore, monitoring its polarized emission, and analyzing changes in the resulting anisotropy. Fluorescence emission intensities are captured by an electron-multiplying charge-coupled device camera. Analyzing four fluorescence intensities emerging from two carefully selected polarized beam splitters enables complete definition of the position of the structural element. Data analysis software detects event transitions with a change point detection algorithm, and also identifies conformational states. These measures yield angstrom-resolution of protein conformational states and track changes in conformation in real-time. This technique is expected to enable monitoring of any rigid structural element in a protein.

 

Caption: Conformational changes of an α-helix (blue) are determined using changes in dipole orientation of an attached fluorophore (green). As the dipole electric field (E) and the vector representing the α-helix (R) are parallel, calculating the changes in E enables determination of the conformational changes of the α-helix.

 

Applications

Microscopic detection of conformational changes of proteins

 

Advantages

•       No longer forced to choose between real-time and high-resolution data

•       Any rigid element of secondary protein structure may be studied

  

 

Stage of Development

Proof-of concept accomplished in laboratory setting
Software under development

 

Desired partnerships

• License

Co-development

 

  

Docket # 19-8726


Patent Information:
For Information, Contact:
Jessica Casciano
University of Pennsylvania
casciano@upenn.edu
Inventors:
Zhe Lu
John Lewis
Keywords: