Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction.

Area of Science:

• Structural biology
• Biochemistry
• Crystallography

Background:

• Pyridoxal 5 -phosphate (PLP) is a vital cofactor for numerous enzymes involved in amino acid metabolism.
• Understanding the reaction diversity of PLP-dependent enzymes requires atomic-level structural insights, particularly concerning hydrogen positions and protonation states.

Purpose of the Study:

• To develop a method for growing large, well-ordered crystals of perdeuterated tryptophan synthase (TS) suitable for neutron diffraction.
• To assess the efficacy of the Toledo Crystallization Box (TCB) for microgravity-based crystal growth and neutron diffraction data collection.

Main Methods:

• Development and implementation of the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device.
• Crystal growth experiments of perdeuterated TS in microgravity aboard the International Space Station (ISS) on Missions Protein Crystal Growth (PCG)-8 and PCG-15.
• X-ray and neutron diffraction data collection and analysis.

Main Results:

• The TCB improved X-ray diffraction and mosaicity for TS crystals grown in microgravity (PCG-8).
• Microgravity-grown TS crystals from PCG-15 yielded superior neutron diffraction data compared to ground controls.
• High-resolution (2.1 Å) neutron diffraction data was obtained from microgravity-grown, perdeuterated TS crystals.

Conclusions:

• Microgravity crystal growth using the TCB is effective for producing high-quality crystals of PLP-dependent enzymes like TS.
• This approach facilitates detailed structural studies, including hydrogen atom positioning, essential for understanding enzyme mechanisms.
• The TCB and microgravity provide a viable pathway for advancing neutron diffraction studies of biologically important molecules.