The overall goal of this component is to develop and test new alignment medium for dipolar coupling measurement and new strategies for obtaining structural restraints in large proteins and protein complexes. In the first phase of this component, we will focus on developing DMA nanostructures for weakly orienting protein complexes that are solubilized in harsh buffer conditions. Residual dipolar couplings (RDCs) are crucial experimental restraints in structure determination of large proteins when long-range NOEs are insufficient. RDCs are also very useful in orienting domains within protein complexes. Based on our initial success in making DNA nanotubes that can form stable liquid crystal in the presence of high concentration of detergents, we will establish a robust protocol for aligning detergent-protein complexes. The membrane protein systems to be tested include a number of ~30 kDa a helical, polytopic membrane proteins. We will also test the alignment of the EIF4e-4g complex, an important translation initiation interaction previously studied by Wagner and colleagues. This protein complex can only reach NMR-feasible concentration in the presence of a detergent known as CHAPS. In the second phase, we will develop methods of measuring structural restraints for large helical proteins. Hydrogen bond length has very small variation and serves as extremely stringent distance constraint for defining secondary structures. Measurement of the small 3JNCg couplings is perhaps the most straightforward and unambiguous method for determining protein sidechain xi angles. We will implement the relaxation optimized pulse elements (ROPE) to enable measurement of 3hJNc'across hydrogen bonds and 3JNCg that defines sidechain rotamer for large deuterated proteins. We will also develop 4D NOESY and 13C detection scheme for resolving resonance overlap. For helical proteins larger than 300 amino acids, it is usually not possible to resolve most of the short-range NOEs in the conventional15N-separated NOESY due to strong overlap of amide resonances. However, they can be resolved in a 4D 15N- and 13C'- edited NOESY (NOESY- HNCO). We will work with Jeff Hoch's group to significantly shorten the experimental time of 4D NOESY- HNCO by implementing nonuniform sampling and maximum entropy reconstruction methods so that it can be of practical use. We will also develop 13C-observation strategies for resolving resonance overlap and apply them to the measurements of 15N relaxation rates and RDCs.
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