Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark.
Chemistry Department, Princeton University, Princeton, NJ 08544, USA.
Acta Crystallogr D Struct Biol. 2021 Apr 1;77(Pt 4):471-485. doi: 10.1107/S2059798321001078. Epub 2021 Mar 30.
Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.
将小分子浸泡到蛋白质晶体的溶剂通道中，是获得与底物或抑制剂等配体结合的晶体复合物的最常见方法。一些蛋白质晶体的溶剂通道足够大，可以允许大分子的掺入，但蛋白质客体到蛋白质晶体中的浸泡尚未被报道。这样的蛋白质主体晶体（此处称为宿主晶体）结合了客体蛋白质，可能在生物技术的广泛应用中有用，例如作为货物系统或用于类似于晶体海绵方法的衍射研究。本研究利用大肠杆菌色氨酸阻遏蛋白（ds-TrpR）的晶体，这些晶体广泛发生结构域交换，适合通过扩散掺入客体蛋白，因为它们坚固且具有大的溶剂通道。共焦荧光显微镜用于跟踪细胞色素 c 和荧光标记的钙调蛋白进入 ds-TrpR 晶体的溶剂通道中的迁移。客体蛋白在数周内均匀分布在晶体中，并在溶剂通道中富集。对含有高浓度掺入客体的宿主晶体进行的 X 射线衍射研究表明，仍可以达到约 2.5Å 的衍射极限。在溶剂通道中观察到微弱的电子密度，但无法通过常规晶体学方法确定客体蛋白结构。讨论了增加客体在宿主晶体中有序性的附加方法，这些方法可能支持未来使用宿主体系进行蛋白质结构测定。该宿主体系在不需要客体结晶有序性的生物技术应用中也可能有用。
