Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Area of Science:

• Structural Biology
• Protein Crystallography
• Biochemistry

Background:

• Tumor necrosis factor (TNF) is a crucial cytokine involved in inflammation and immune responses.
• Understanding TNF's molecular structure is vital for developing targeted therapies.

Purpose of the Study:

• To obtain and characterize crystalline forms of tumor necrosis factor (TNF).
• To elucidate the oligomeric state of TNF in solution through crystallographic analysis.

Main Methods:

• Protein crystallization using ammonium sulfite and magnesium sulfate solutions.
• Isoelectric focusing under native and denaturing conditions.
• X-ray diffraction analysis of crystal structures.

Main Results:

• Two distinct crystal forms of TNF were obtained: rhombohedral and tetragonal.
• Isoelectric focusing confirmed TNF exists as trimers in solution.
• Rhombohedral crystals (space group R3) revealed a 51,000-dalton trimer per unit cell and diffracted to 1.85 A.
• Tetragonal crystals (space groups P4(3)2(1)2 or P4(1)2(1)2) also contain a trimer in the asymmetric unit and diffracted beyond 3 A.

Conclusions:

• Crystallization of TNF in multiple forms provides valuable structural data.
• The trimeric state of TNF in solution is confirmed, crucial for its biological function.
• These crystal structures offer a foundation for understanding TNF's interactions and for drug design.