A circumventing role for the non-native intermediate in the folding of β-lactoglobulin.

Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G ̅o model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine β-lactoglobulin (βLG). This protein has an intermediate with a non-native α-helical structure, although its native form is predominantly composed of β-structure. In this study, we prepared mutants whose α-helical and β-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose β-sheet propensity was increased, showed a folding intermediate with an amount of β-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high α-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.