Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane

Area of Science:

• Biophysics
• Molecular Imaging
• Cell Biology

Background:

• Second harmonic generation (SHG) microscopy offers unique advantages for biological imaging, particularly voltage sensing.
• Genetically-encoded SHG chromophores are underdeveloped, limiting applications.
• SHG signal generation requires non-centrosymmetric media, necessitating anisotropic chromophore arrangements.

Purpose of the Study:

• To engineer a genetically-encoded chromophore capable of producing strong SHG signals.
• To achieve asymmetric chromophore arrangements at the plasma membrane-cytoplasm interface for SHG imaging.
• To explore the potential of engineered proteins for voltage imaging applications.

Main Methods:

• Engineered the fluorescent protein mVenus by adding a farnesylation motif and modifying its surface to create an electrostatic patch (mVe9Knus-CVIM).
• Investigated the targeting of mVe9Knus-CVIM to the plasma membrane in HEK293 cells.
• Measured SHG signals and characterized the orientation of molecular hyperpolarizability relative to the membrane normal.

Main Results:

• mVe9Knus-CVIM was efficiently and geometrically targeted to the plasma membrane.
• The engineered protein exhibited measurable SHG signals in HEK293 cells.
• The hyperpolarizability of mVe9Knus-CVIM was found to be oriented at a small angle (~7.3°) from the membrane normal, consistent with design principles.

Conclusions:

• Genetically-encoded SHG chromophores can be engineered for specific cellular localization and anisotropic arrangements.
• mVe9Knus-CVIM demonstrates the feasibility of using engineered proteins as SHG probes.
• This molecular platform holds promise for advanced imaging techniques, including membrane potential imaging.