Lanthanide binding and IgG affinity construct: potential applications in solution NMR, MRI, and luminescence...

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Paramagnetic lanthanide ions are valuable tools for protein structural studies using NMR, MRI, and optical microscopy.
• Many proteins lack native metal-binding sites, necessitating protein engineering for lanthanide ion incorporation.
• Developing chimeric proteins that bind lanthanide ions while maintaining target affinity is a significant challenge.

Purpose of the Study:

• To characterize a redesigned immunoglobulin G-binding protein (Z-L2LBT) engineered with a lanthanide-binding motif.
• To assess the utility of lanthanide-bound Z-L2LBT for structural and dynamic investigations.
• To evaluate Z-L2LBT as a novel reagent for molecular imaging.

Main Methods:

• Protein engineering of an immunoglobulin G-binding protein to incorporate a lanthanide-binding motif.
• Characterization of terbium (Tb³⁺) binding affinity to the engineered protein (Z-L2LBT).
• Utilizing paramagnetic effects of various lanthanide ions (Dy³⁺, Yb³⁺, Ce³⁺) in NMR spectroscopy.
• Developing Z-L2LBT complexes with gadolinium (Gd³⁺) and Tb³⁺ for imaging applications.

Main Results:

• The engineered Z-L2LBT protein demonstrated high affinity (130 nM) for Tb³⁺.
• Lanthanide ions induced paramagnetic effects, enabling the determination of a structural model for metal-complexed Z-L2LBT.
• Preliminary characterization of IgG Fc glycan positions was achieved using NMR.
• Z-L2LBT:Gd³⁺ and Z-L2LBT: Tb³⁺ complexes showed promise as reagents for MRI and luminescence microscopy, respectively.

Conclusions:

• Engineered Z-L2LBT protein effectively binds lanthanide ions, serving as a versatile tool for structural biology.
• The protein facilitates advanced NMR-based structural and dynamic analyses.
• Z-L2LBT represents a novel platform for developing lanthanide-based imaging probes.