Thermal behavior of proteins: heat-resistant proteins and their heat-induced secondary structural changes.

Most proteins are denatured by heat treatment, and the process is usually irreversible. However, some proteins, such as hyperthermophilic proteins are known to be stable even at the boiling temperature of water. We here describe a systematic investigation of thermal behavior of proteins by purifying and characterizing some heat-resistant proteins (HRPs) that are not aggregated upon heat treatment. Although most proteins were precipitated by boiling in a water bath, about 20 and 70 wt % of total proteins appeared to be heat-resistant in Jurkat T-cell lysates and human serum, respectively. We identified major HRPs from Jurkat T-cells and human serum by N-terminal amino acid sequencing and Western blot analysis. HRPs of 20 and 45 kDa (HRP20 and HRP45) were identified as alpha-synuclein and calreticulin, respectively, and HRPs of 60, 27, and 16 kDa (HRP60, HRP27, and HRP16) were identified as human serum fetuin, apolipoprotein A-I, and transthyretin, respectively. By a systematic investigation of the effect of heat on the secondary structure of the purified HRPs by circular dichroic spectroscopy, we observed four major types of thermal behavior, suggesting that the proteins could protect themselves through these pathways. Although our analysis is restricted to protein secondary structural changes, our data indicate that heat resistance of protein can be achieved in several different ways depending on the thermodynamic stability of native (N), unfolded (U), denatured (D), and intermediate (I) states.