Uniform expression and isotopic enrichment of multiple constructs of the fusion domain of hemagglutinin has been achieved, resulting in samples that are reasonably homogeneous (>70%) and suitable for NMR structural studies. Prior work on this system, primarily by Tamm and co-workers (U. of Virginia) was based primarily on NOE data and yielded relatively low resolution data without a clear view of the structural transition underlying the switch to fusion. To enhance the resolution, we have recorded extensive residual dipolar coupling measurements in two different alignment media (polyacrylamide gel and liquid crystalline DNA) for both N-H and Ca-Ha interactions. Structures derived from the new data for the monomeric form of the fusion domain show many similarities but also distinct differences relative to those of earlier studies. The high pH data provide evidence for the presence of both a helical and a small fraction of beta-sheet form for the fusion domain, complicating the analysis of NOE data, and making it essential to guide the structure determination by novel methods, based on chemical shifts and RDCs that report exclusively on the major component in the system. Paramagnetic relaxation agents are used for probing the embedding of the fusion domain in the lipophilic environment of the micelle, which is found to depend significantly on pH, as does the kink in the center of the helical fusion domain.
