Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the 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 (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization 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. The conclusion helps to explain how the molten globule state acquires its structure and stability. Study holds ProTherm entries: 7916, 7917 Extra Details: N->I cooperative stages; intermediate; ring-current shifts;,aromatic cluster
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||4|
|Proteins||Lysozyme C, milk isozyme ; Lysozyme C, milk isozyme|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence SKIFSKCELARKLKSMGMDGFHGYSLANWVCMAEYESNFNTQAFNGRNSNGSSDYGIFQLNSKWWCKSNSHSSANACNIMCSKFLDDNIDDDIACAKRVVKDPNGMSAWVAWVKHCKGKDLSKYLASCNL|