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Current Research Summaries

  • [Mini review] Structural aspects of EF-hand containing calcium buffers, sensors, and S100 family

    The EF-hand motif is one of the common calcium binding motifs, and the protein family containing this motif plays an essential role in the regulating all aspects of cellular processes. The EF-hand motif is a helix-loop-helix structure and the canonical EF-hand chelates Ca2+ by seven ligands in the loop with pentagonal bipyramid geometry. Many high-resolution structures of EF-hand containing calcium binding proteins (CaBPS), as well as the complex with target proteins, have been identified. They exhibit a various loop composition and structure and they affect a great diversity of target interaction and functions. In this review, structural details of the EF-hand motif are discussed to understand the Ca2+ binding mechanism, Ca2+-induced conformational change in EF-hand CaBPS, and the diversity of their complex with their target molecules.

  • [Mini review] Application of helix fusion method in structural biology

    Generating artificial protein assemblies with complex shapes requires a method of connecting protein components with stable and predictable structures. Because they have uniform structures, alpha helices can provide an excellent linker for connecting proteins with predictable structures. However, except for a few exceptional cases, early attempts to ligate two proteins by fusion of terminal alpha helices were not successful. In order to solve this problem, several new methods have been developed in recent years. In the chemical cross-linker method, the linker helix is stabilized by a chemical cross-linker that can force an alpha helical geometry by fixing the distance between two cysteine residues. In the shared-helix method, the linker helix is generated by overlapping pairs of alpha helices by 1~2 turns using a molecular modeling program. The amino acid sequence at the overlapped site is chosen from the two natural sequences that would stabilize the alpha helical linker. These two helix fusion methods are expected to be useful in structural biology because they can enhance the crystallization property of challenging target proteins by providing a rigid and crystallizable surface. They also can be used to produce artificial protein complexes by connecting the target protein to a large backbone protein. The resulting protein complex effectively increases the size of the target protein for cryo-electron microscopy study. In this review, we summarize recent progress of the helix fusion methods and their application to structural study of challenging proteins.

  • [Mini review] Time-resolved serial femtosecond X-ray crystallography

    Time-resolved serial femtosecond crystallography (TR-SFX) with X-ray free electron lasers (XFELs) is a powerful new technique for the study of protein dynamics on unprecedented time scales, at room temperature, without structureaffecting radiation damage. The construction of Pohang Accelerator Laboratory XFEL, a 0.1-nm hard X-ray free-electron laser facility based on a 10-GeV S-band linear accelerator in Pohang, Korea, provides a great opportunity to exploit and develop this novel methodology for structural biological studies. This review summarizes the state of the art in TR-SFX including key contributions of pump-probe and mix-and-inject TR-SFX to the field of protein dynamics.

  • [Crystallization] Crystallization and preliminary X-ray diffraction analysis of a toxin-antitoxin MazEF complex from the extremophile Deinococcus radiodurans

    Toxin–antitoxin (TA) systems are ubiquitous among most of prokaryotes and govern the cell death or growth arrest in response to environmental cues. TA systems are associated with adaptation of pathogens to unfavorable environments, indicating their potential as a target for antibiotics. Here, we purified and crystallized the TA complex from the extremophile Deinococcus radiodurans. The TA complex (DrMazEF) was co-expressed and pulled using the N-terminal glutathione S-transferase-tagged DrMazF. The complex was crystallized in 100 mM citric acid pH 3.5 containing 25% PEG3350. The crystal diffracted X-ray to a 2.6 Å resolution and belonged to the space group P212121, with the unit cell parameters a = 46.01, b = 74.04, and c = 138.26 Å. The asymmetric unit of the crystal had six molecules in two heterotrimeric complexes with a calculated Mathew’s coefficient of 1.84 Å3 Da-1 and a solvent content of 33.18%.

  • [Crystallization] Crystallization and preliminary diffraction analysis of dual specificity phosphatase 13a

    Dual specificity phosphatases (DUSPs) include MAP kinase phosphatases and atypical dual specificity phosphatases, and mediate cell growth and differentiation. They are considered as drug targets against cancers, diabetes, immune diseases, and neuronal diseases. Two different DUSPs, DUSP13a and DUSP13b are coded in one gene (DUSP13) whose alternative splicing results in two sequence-related proteins with different target specificities. The crystal structure of DUSP13b showed a canonical DUSP fold. However, the structure of DUSP13a has not been determined yet. To understand structural mechanism of distinctive target specificities of sequence-related DUSPs, we prepared diffraction-quality crystals of DUSP13a. Cysteine residues in DUSP13a gene were mutated to serine or alanine to prevent cysteine oxidation. From the stabilized protein, we were able to grow good crystals that diffracted to 1.7 Å resolution. The preliminary diffraction analysis revealed that the crystal is in the space group P21 with unit cell parameters of a = 40.04 Å, b = 89.34 Å, c = 45.90 Å, α = 90.00°, β = 89.87° and γ = 90.00°.