Protein stability measurement is essential to study the relationship between the structure and the function of proteins. Protein stability is usually estimated by linear extrapolation method (LEM) analysis of denaturant-induced unfolding measurements. Through LEM, the native stability of protein (￥?GoNU) is obtained. Furthermore, LEM provides the m-value which is known to reflect the change in the solvent accessible surface area (SASA) when a protein unfolds. The m-values are normally observed constant upon mutation or change in solvent conditions. However, the m-value changes have been observed in several experiments. It has been interpreted that the change in m-value is due to; (1) change in the structure of the native or unfolded state, or (2) accumulation of folding intermediates which reduces the cooperativity of folding reaction. The change in m-value was observed in the unfolding measurements of hydrophobic core variant ubiquitin, HubWA. The unfolding measurements of ubiquitin were normally performed at pH 5. At this solvent pH, HubWA observed to follow a two-state folding mechanism. However, unfolding of HubWA observed by far-UV circular dichroism, a spectral probe that measures secondary structural change, showed that the m-value at pH 7 and 9 are significantly smaller than that observed at pH 5. Based on the unfolding of HubWA observed by fluorescence spectroscopy, a spectral probe that measures tertiary structural change, the decrease in m-value at pH 9 is considered due to the change in structure of the native state, while the decrease in m-value at pH 7 is considered due to the accumulation of folding intermediates.
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