Influence of divalent cations in protein crystallization

Area of Science:

• Structural biology
• Biochemistry
• Crystallography

Background:

• Leucine/isoleucine/valine-binding protein (LIVBP) and leucine-specific binding protein (LBP) are crucial periplasmic receptors in bacterial active transport systems.
• Crystallization of ligand-bound forms of LIVBP and LBP has been challenging for approximately 15 years.
• Understanding the structure of these proteins is vital for deciphering nutrient transport mechanisms.

Purpose of the Study:

• To identify cations that enhance the crystallization of ligand-bound LIVBP and LBP.
• To optimize crystal growth conditions for improved diffraction quality.
• To facilitate high-resolution structural determination of these essential bacterial proteins.

Main Methods:

• Systematic screening of various divalent cations as additives during protein crystallization.
• Utilizing polyethylene glycol 8000 as a precipitant for LIVBP crystallization.
• Optimizing cation concentration to achieve desired crystal morphology and diffraction.

Main Results:

• Cadmium ions (Cd2+) dramatically improved crystal size, morphology, and diffraction for both LIVBP and LBP.
• Cd2+ yielded large hexagonal prism crystals of LIVBP, while other cations often resulted in needle-shaped crystals.
• Optimal Cd2+ concentrations were 1 mM for LIVBP and 100 mM for LBP, yielding crystals diffracting to at least 1.7 Å.

Conclusions:

• Cd2+ is a highly effective additive for crystallizing ligand-bound LIVBP and LBP.
• The optimal concentration of Cd2+ varies significantly between LIVBP and LBP.
• This work provides a breakthrough for the structural study of these important bacterial transport proteins.