Thioredoxin as a fusion tag for carrier-driven crystallization

Area of Science:

• Structural Biology
• Protein Crystallography
• Molecular Biology

Background:

• Obtaining diffracting crystals for structural studies is often challenging.
• Fusion proteins can aid in the crystallization of difficult targets.

Purpose of the Study:

• To determine the crystal structure of the U2AF homology motif (UHM) domain of splicing factor Puf60.
• To investigate the role of fusion partners and linker sequences in protein crystallization.

Main Methods:

• Crystallization and X-ray diffraction of a fusion protein comprising thioredoxin A and the Puf60-UHM domain.
• Comparison of two linker sequences (GSAM and GSPPM) between the fusion partners.
• Nuclear Magnetic Resonance (NMR) relaxation studies to assess interdomain mobility.

Main Results:

• The fusion protein with the GSAM linker crystallized, yielding a well-defined structure.
• The GSPPM linker resulted in a more flexible fusion protein that did not yield diffracting crystals.
• NMR data indicated reduced interdomain mobility for the GSAM-linked fusion protein.

Conclusions:

• Thioredoxin A is an effective fusion partner for crystallizing challenging proteins.
• Short, specific linker sequences (e.g., GSAM) are crucial for successful crystallization.
• Linker length and flexibility significantly impact the ability to obtain diffracting crystals.