Nuclear Magnetic Resonance--new Methods And Molecular St

核磁共振--新方法与分子研究

• 批准号: 6810190
• 负责人: Ad - Bax
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6810190
• 关键词: biomagnetism measurement; calcium ion; calmodulin; chemical binding; intermolecular interaction; liquid crystal; magnetism; method development; micelles; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure; rhodopsin; structural biology

We have extended our technology for studying macromolecular structure in solution under weakly aligning conditions. New developments increase both the size of systems that can be studied, and the accuracy that can be obtained. Rhodopsin-containing membranes contain sufficient residual diamagnetic susceptibility anisotropy that they align spontaneously in a sufficiently strong magnetic field. Undeca-peptide analogs of the C-terminus of the gamma-subunit of transducin are known to bind weakly but specifically to photo-activated rhodopsin. The orientation of the peptide occurring upon binding to rhodopsin, following photo-activation, was used by us to provide precise structural and orientational information on the peptide in the bound state, and thereby on the orientation of the entire G protein in the receptor-bound state. A prerequisite for such studies is a relatively low affinity of the peptide for the membrane-anchored receptor. For water-soluble proteins and nucleic acids, weak alignment allows very accurate measurement of internuclear dipolar couplings, thereby providing extremely precise information on local as well as global structure. For a small, 56-residue domain, we used this technology to study planarity of peptide groups and found that the out-of-plane position of the amide hydrogen has a root-mean-square value of less than about "4", much smaller than predicted on the basis of literature theoretical calculations, but in good agreement with neutron diffraction results. The in-plane deviation from the line bisecting the C?-N-Ca angle is less than "2". For CO-ligated adult human hemoglobin, analogous technology revealed that in solution, at 100 mM ionic strength, the quaternary structure is about half-way intermediate between the so-called R and R2 states. Application to nucleic acids shows that accurate measurement of the bending of the helical axis is possible with the weak alignment technology. We also have succeeded in measurement of interactions over much larger distances, up to 12 ?, than previously possible by NMR.

我们扩展了在弱排列条件下研究溶液中大分子结构的技术。新的发展既增加了可研究系统的规模，也增加了可获得的准确性。含视紫红质的膜含有足够的残余抗磁化率各向异性，使得它们在足够强的磁场中自发排列。已知转导蛋白γ亚基C末端的十一肽类似物与光激活视紫红质微弱但特异性结合。我们利用光激活后与视紫红质结合时肽的方向来提供肽在结合状态下的精确结构和方向信息，从而提供整个 G 蛋白在受体结合状态下的方向信息。此类研究的先决条件是肽与膜锚定受体的亲和力相对较低。对于水溶性蛋白质和核酸，弱比对可以非常准确地测量核间偶极耦合，从而提供有关局部和整体结构的极其精确的信息。对于一个56个残基的小结构域，我们利用该技术研究了肽基团的平面性，发现酰胺氢的面外位置的均方根值小于约“4”，比根据文献理论计算预测的要小得多，但与中子衍射结果非常吻合。距Cα-N-Ca角二等分线的面内偏差小于“2”。对于 CO 连接的成人血红蛋白，类似技术表明，在溶液中，在 100 mM 离子强度下，四级结构大约处于所谓的 R 和 R2 状态之间的中间位置。核酸的应用表明，利用弱对准技术可以精确测量螺旋轴的弯曲度。我们还成功地测量了比以前通过 NMR 实现的更远距离（高达 12 Ω）的相互作用。