Nuclear Magnetic Resonance--new Methods And Molecular St

我们已经扩展了我们的技术，用于研究弱排列条件下溶液中的大分子结构。新的发展既增加了可以研究的系统的规模，也增加了可以获得的精度。含有视紫红质的膜含有足够的残余抗磁各向异性，使它们在足够强的磁场中自发对准。已知的转导蛋白伽马亚单位C末端的十一肽类似物与光激活的视紫红质结合很弱，但具有特异性。在光激活后，当与视紫红质结合时，我们使用多肽的取向来提供处于结合状态的肽的精确结构和取向信息，从而提供处于受体结合状态的整个G蛋白的取向。这类研究的先决条件是多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偶联，从而提供关于局部和全球结构的极其精确的信息。对于一个小的，56个残基的区域，我们用这项技术研究了多肽基团的平面性，发现酰胺氢的平面外位置的均方根值小于“4”，比文献理论计算预测的要小得多，但与中子衍射结果很好地吻合。与C？-N-Ca角等分线的面内偏差小于“2”。对于共连接的成人血红蛋白，类似的技术表明，在溶液中，在100 mM的离子强度下，四元结构大约是所谓的R和R2状态之间的中间状态。对核酸的应用表明，利用弱排列技术可以精确测量螺旋轴的弯曲。我们还成功地测量了比以前核磁共振可能的更大距离的相互作用，最大可达12？

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.