Warkentin Matthew, Thorne Robert E
Physics Department, Cornell University, Ithaca, NY 14853, USA.
J Appl Crystallogr. 2009 Oct 1;42(Pt 5):944-952. doi: 10.1107/S0021889809023553. Epub 2009 Aug 1.
Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s(-1) - 10(3)-10(4) times slower than in conventional flash cooling - while continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2 degrees . Most of the mosaicity increase occurs above approximately 200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3-6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.
无冷冻保护剂的奇异果甜蛋白晶体已以0.1 K s⁻¹的速率从300 K冷却至100 K，比传统的快速冷却慢10³ - 10⁴倍，同时持续收集X射线衍射数据，以便追踪冷却过程中蛋白质晶格和溶剂性质的演变。衍射图谱在任何温度下均未显示出结晶冰的迹象。这表明蛋白质分子的晶格本身就是一种出色的冷冻保护剂，并且从1.5 M母液中掺入酒石酸钠钾后，冰核形成速率至少与70%甘油溶液中的一样低。缓慢冷却过程中晶体质量保持较高，在100 K时平均镶嵌度为0.2°。大部分镶嵌度增加发生在大约200 K以上，此时溶剂仍为液态，并且与晶胞的各向异性收缩同时发生。在接近180 K时，溶剂行为转变为类似固体的行为，进一步冷却时晶体有序度没有额外的降解。B因子随温度的变化清楚地表明在210 K附近存在蛋白质动力学转变，并且在较低温度下，斜率dB/dT比任何其他蛋白质报道的小3 - 6倍。这些结果确立了在300 K至100 K之间对蛋白质结构和动力学进行完全温度控制研究可行性。
