Probing site-specific calmodulin calcium and lanthanide affinity by grafting

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Calmodulin (CaM) is crucial for calcium (Ca2+) signaling, acting as a sensor and regulator.
• Understanding site-specific Ca2+ binding in EF-hand proteins like CaM is challenging.
• Ca2+ binding kinetics and affinities are vital for CaM's biological functions.

Purpose of the Study:

• To determine the intrinsic Ca2+ affinities of the four EF-hand loops (I-IV) of calmodulin.
• To investigate the determinants of relative Ca2+ binding affinities in CaM's EF-hand loops.
• To establish a new method for site-specific analysis of Ca2+ binding properties.

Main Methods:

• Grafting individual CaM EF-hand loops into a host protein (CD2 domain 1).
• Utilizing tryptophan residues in the host protein for fluorescence-based assays.
• Employing Tb3+ aromatic resonance energy transfer to measure metal-binding affinities (Tb3+, Ca2+, La3+).

Main Results:

• EF-loop I exhibited the strongest binding affinity for Ca2+, La3+, and Tb3+.
• EF-loop IV demonstrated the weakest metal-binding affinity among the four loops.
• Dissociation constants for Ca2+ were determined: Loop I (34 µM) > Loop III (185 µM) ≈ Loop II (245 µM) > Loop IV (814 µM).

Conclusions:

• Ca2+ binding affinities are governed by the number of negatively charged ligand residues and electrostatic repulsion.
• The findings support a charge-ligand-balanced model for EF-hand protein Ca2+ binding.
• The novel grafting strategy enables site-specific characterization of Ca2+ binding and cooperativity in CaM.