STRUCTURAL STUDY OF MURINE GAMMA-CRYSTALLIN BY COMBINED USE OF SAXS AND WEAK ALI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。使用一种称为分子片段置换(MFR)的过程，即使在完全没有NOE或二面体信息的情况下，也可以通过仅使用残留偶极耦合(RDC)和化学位移的核磁共振来快速求解蛋白质结构。将这项技术应用于小鼠G-晶体蛋白，得到了其两个球状结构域的结构，与以前研究的其他G-晶体蛋白的X射线结构很好地一致。虽然偶极耦合信息对两个g-晶体蛋白结构域的相对取向提供了非常严格的限制，但核磁共振数据缺乏翻译信息。模拟表明，SAXS数据将产生这一关键信息。需要高质量的小鼠g-晶体蛋白的SAXS实验数据来证明SAXS和弱排列核磁共振的结合为快速确定大分子及其络合物的结构提供了一种新的强有力的工具。由于球状结构域的结构经常是事先已知的，它们的相对取向很好地由从弱序列核磁共振获得的偶极耦合来确定，高信噪比的SAXS数据(在S/N比至少为3：1，0.5时获得)提供了快速解决多模块蛋白质以及蛋白质-蛋白质和蛋白质-核酸复合体结构所需的翻译信息。如果在该系统上取得成功，该方法可以应用于溶液中的多种模块化或多结构域蛋白质，从而提供对各种条件和功能状态下的结构域相互作用的洞察。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Using a procedure termed molecular fragment replacement (MFR) it has become possible to rapidly solve protein structures by NMR using only residual dipolar couplings (RDCs) and chemical shifts, even in the complete absence of NOE or dihedral information. Application of this technology to murine g-crystallin yields structures for its two globular domains that are in good agreement with x-ray structures of other previously studied g-crystallins. Although the dipolar coupling information provides very tight restraints for the relative orientation of the two g-crystallin domains, the NMR data lack translational information. Simulations indicate that the SAXS data will yield this critical information. High quality experimental SAXS data on murine g-crystallin is needed to demonstrate that the combination of SAXS and weak alignment NMR provides a new and powerful tool for rapidly determining structures of macromolecules and their complexes. With the structure of globular domains frequently known in advance, and their relative orientation well determined by the dipolar couplings obtained from weak alignment NMR, high signal-to-noise SAXS data (obtained at a S/N ratio of at least ca 3:1 at 0.5/¿¿, provides the translational information needed to rapidly solve the structure of multi-module proteins, and protein-protein and protein-nucleic acid complexes. If successful on this system, this approach could be applied to a wide variety of modular or multidomain proteins in solution, providing insights into domain interactions under a variety of conditions and functional states.