Mizuno Toshihisa, Hasegawa Chinatsu, Tanabe Yoichi, Hamajima Kenta, Muto Takashi, Nishi Yoshinori, Oda Masayuki, Kobayashi Yuji, Tanaka Toshiki
Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Nagoya, Aichi, 466-8555, Japan.
Chemistry. 2009;15(6):1491-8. doi: 10.1002/chem.200800855.
The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled-coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand-binding characteristics of the cavities, we originally designed two other coiled-coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size-complementary organic compounds. The dissociation constants, K(d), of AM2W were 220 nM for adamantane, 81 microM for 1-adamantanol, and 294 microM for 1-adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.
从头设计蛋白质中疏水腔的设计与表征提供了有关从头设计蛋白质功能的广泛信息。我们设计了一种具有疏水口袋状腔的从头四聚体卷曲螺旋蛋白。此前已有报道具有疏水腔的四聚体卷曲螺旋。通过将α位置的一个亮氨酸残基替换为丙氨酸，可靠地创建了不会因肽链松散而变平的疏水腔。为了详细研究这些腔的配体结合特性，我们最初设计了另外两种卷曲螺旋蛋白：AM2，在束状结构中心的相邻α和δ位置有八个丙氨酸取代；AM2W，在相同位置有一个色氨酸和七个丙氨酸取代。为了增强螺旋肽的缔合，每个螺旋肽都用柔性接头连接，从而形成单肽链。尽管疏水堆积减弱，但这些蛋白质呈现出对应于超螺旋结构的圆二色光谱。AM2W对尺寸互补的有机化合物表现出结合亲和力。通过荧光滴定分析测定，AM2W对金刚烷的解离常数K(d)为220 nM，对1-金刚烷醇为81 μM，对1-金刚烷乙酸为294 μM。尽管与预期相反，但AM2没有表现出任何结合亲和力，这可能是由于设计的疏水腔周围存在结构缺陷。有趣的是，AM2W通过配体结合表现出结构稳定性增加。用有机配体填补结构缺陷有望促进蛋白质折叠。
