Analysis on the early events of protein folding at subzero temperatures

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Protein takes (a) definite pathway(s) when it folds into its native form. It is so-called folding problem. One of the most important subject in the folding process is when and how the three major conformational formation takes place ; i.e. secondary structure formation, compaction and formation of the folding core.Protein folding starts at the time range of sub-micro-seconds. We reduced this rate to millisecond region, by decreasing temperature so as to slow down the folding process. With the use of stopped-flow technique combined with circular dichroism, fluorescence and X-ray scattering, we could monitor formation of alpha-helix, folding core formation and compaction. Results so far obtained are ; (1) formation rate of alpha-helix is too fast to be monitored even at -38C. (2) In case of bovine beta-lactogloblulin, we monitored a phase of alpha-helix increase at -28C. (3) In case of ubiquitin, the initial burs phase of alpha-helical formation was observed at -20C.From the findings above, it would be suggested that formation of alpha-helix is commonly observed at the early stage of the folding even in the case of beta-rich proteins.Results above are also very consistent with findings observed by temperature-jump method, and thus demonstrate the importance of our methodology ; cooling down the folding process.