Suzuki Nobuhiro, Hiraki Masahiko, Yamada Yusuke, Matsugaki Naohiro, Igarashi Noriyuki, Kato Ryuichi, Dikic Ivan, Drew David, Iwata So, Wakatsuki Soichi, Kawasaki Masato
Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki 305-0801, Japan.
Acta Crystallogr D Biol Crystallogr. 2010 Oct;66(Pt 10):1059-66. doi: 10.1107/S0907444910032944. Epub 2010 Sep 18.
The generation of crystal lattice contacts by proteinaceous tags fused to target proteins is an attractive approach to aid in the crystallization of otherwise intractable proteins. Here, the use of green fluorescent protein (GFP) fusions for this purpose is demonstrated, using ubiquitin and the ubiquitin-binding motif (UBM) of Y-family polymerase ι as examples. The structure of the GFP-ubiquitin fusion protein revealed that the crystal lattice was formed by GFP moieties. Ubiquitin was accommodated in the lattice through interactions with the peripheral loops of GFP. However, in the GFP-UBM fusion crystal UBM formed extensive interactions with GFP and these interactions, together with UBM dimerization, mediated the crystal packing. Interestingly, the tyrosine residues that are involved in mediating crystal contacts in both GFP-ubiquitin and GFP-UBM crystals are arranged in a belt on the surface of the β-barrel structure of GFP. Therefore, it is likely that GFP can assist in the crystallization of small proteins and of protein domains in general.
通过将蛋白质标签融合到目标蛋白上产生晶格接触，是一种有助于使原本难以结晶的蛋白结晶的有吸引力的方法。在此，以泛素和Y家族聚合酶ι的泛素结合基序（UBM）为例，展示了为此目的使用绿色荧光蛋白（GFP）融合体。GFP-泛素融合蛋白的结构表明，晶格是由GFP部分形成的。泛素通过与GFP的外周环相互作用而容纳在晶格中。然而，在GFP-UBM融合晶体中，UBM与GFP形成了广泛的相互作用，并且这些相互作用与UBM二聚化一起介导了晶体堆积。有趣的是，在GFP-泛素和GFP-UBM晶体中参与介导晶体接触的酪氨酸残基，排列在GFP的β-桶状结构表面的一条带上。因此，GFP很可能通常能协助小蛋白和蛋白结构域的结晶。
