Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan.
Biophys J. 2011 Oct 19;101(8):2037-42. doi: 10.1016/j.bpj.2011.09.004.
We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D(2)O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.
我们开发了一种新颖的、据我们所知的技术，用于实时监测同寡聚体蛋白质中的亚基交换，使用氘辅助小角中子散射（SANS），并将其应用于蛋白酶体 α7 亚基的十四聚体。在 81% D2O 溶液中，同位素正常和氘代的十四聚体表现出相同的 SANS 图谱。混合这些溶液后，低 q 区域的同位素敏感 SANS 强度逐渐降低，表明亚基交换，而小角 X 射线散射谱保持不变，证实了交换过程中十四聚体颗粒的结构完整性。零角散射强度的动力学分析表明，1）每个十四聚体中只有两个亚基被交换，2）交换过程至少涉及两个步骤。这项研究强调了氘辅助 SANS 的有用性，它不仅可以提供同寡聚体蛋白质的分子大小和形状的定量信息，还可以提供其动力学性质的定量信息。
