Self-association of lysozyme as seen by magnetic relaxation dispersion

The pH and salt dependent self-association of hen egg-white lysozyme (HEWL) has been studied extensively, mainly by scattering techniques, but it has proven difficult to distinguish oligomerization from long-range interactions. Because HEWL is the principal model system in studies of protein crystallization, it is important to establish its oligomerization behavior unambiguously. Here, we address this problem with the aid of proton magnetic relaxation dispersion (MRD), which can determine the sizes and populations of coexisting oligomers with minimal influence of long-range interactions. We find that HEWL is monomeric at pH 4 and dimeric at pH 9. Dimers as well as a higher oligomer are formed also at pH 4 in the presence of K2SO4. The dimer observed in solution has the same rotational correlation time as a dimer present in tetragonal HEWL crystals. The higher oligomer, consisting of about 16 HEWL monomers, is more abundant as the saturation limit is approached, but is suppressed by addition of sulfobetaine. It is also promoted by contaminating proteins, present in commercial HEWL preparations. These findings can explain why HEWL tends to form amorphous precipitates in the presence of sulfate and why crystallization of HEWL is facilitated by sulfobetaine. The MRD data also yield a residence time of 2 ns for several protein-associated water molecules, probably including the four-molecule cluster buried between the and subunits of HEWL.