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

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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.