Fluorescence-based biosensing of zinc using carbonic anhydrase

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Measuring free zinc (Zn(II)) and imaging zinc fluxes is challenging due to low concentrations and interference from cations like magnesium (Mg) and calcium (Ca).
• Existing methods face technical difficulties in accurately quantifying zinc levels in biological systems.

Purpose of the Study:

• To develop novel fluorescent zinc indicators with high sensitivity and selectivity.
• To enable quantitative imaging of free zinc levels in biological samples using microscopy.

Main Methods:

• Engineered fluorescent zinc indicators utilizing human apo-carbonic anhydrase II, a protein with high affinity for Zn(II).
• Transduced zinc levels via changes in fluorescence intensity, wavelength ratio, lifetime, and anisotropy.
• Employed site-directed mutagenesis to create variants of carbonic anhydrase with modified properties.

Main Results:

• Developed a series of fluorescent indicators with superb sensitivity and selectivity for Zn(II).
• Achieved quantitative imaging of zinc levels using fluorescence lifetime and anisotropy.
• Generated carbonic anhydrase variants with enhanced zinc affinity, altered selectivity, and improved binding kinetics.

Conclusions:

• Protein-based fluorescent indicators offer a powerful tool for measuring and imaging free zinc.
• Site-directed mutagenesis provides a versatile approach to optimize sensor performance for specific applications.
• These advanced indicators overcome limitations of previous methods for zinc analysis in biological research.