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) over bar 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 beta-lactoglobulin (beta LG). This protein has an intermediate with a non-native alpha-helical structure, although its native form is predominantly composed of beta-structure. In this study, we prepared mutants whose a-helical and beta-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose beta-sheet propensity was increased, showed a folding intermediate with an amount of beta-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high a-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.