Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme

Here, we show that an unfolded intermediate of canine milk lysozyme is extr aordinarily stable compared with that of the other members of the lysozyme- alpha-lactalbumin superfamily, which has been studied previously. The stabi lity of the intermediate of this protein was investigated using calorimetry , CD spectroscopy, and NMR spectroscopy, and the results were interpreted i n terms of the structure revealed by X-ray crystallography at a resolution of 1.85 Angstrom to an R-factor of 17.8%. On the basis of the results of th e thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the H-1 NMR spectra of canine milk lysozyme at 60 degrees C, essentially 10 0% of which exists in the intermediate, showed that small resonance peaks t hat arise from ring-current shifts of aliphatic protons are still present i n the upfield region from 0 to -1 ppm. The protein at this temperature (60 OC) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (AN S) with enhancement of the fluorescence intensity compared with that of nat ive and thermally unfolded states. We interpret that the extraordinarily st able intermediate is a molten globule state, and the extraordinary stabiliz ation of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. T he conclusion helps to explain how the molten globule state acquires its st ructure and stability.