A strategically-designed light microscope setup with data analysis software resolves angstrom-scale protein conformational changes in real time.
Protein conformational changes are intricately involved in their mechanisms of action. This information is key to the 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.
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.
Zhe Lu and John H. Lewis
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.
Microscopic detection of conformational changes of proteins
- 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
Docket # 19-8726