Effects of Labeling Thiophilic FRET Dyes on the Stability and Dimerization Process of β-Lactoglobulin

The stability and dimeric state of β-lactoglobulin (β-lg) can be dramatically affected by labeling the thiophilic agent to Cys121, whereas the underlining mechanism of such an effect is still unclear. We label a fluorescence-resonance-energy-transfer (FRET) pair of donor (1,5-IAEDANS) and acceptor (5-IAF) dyes to Cys121 of β-lg monomers to investigate the effect of bulky thiophilic modification on the structure and stability of β-lg. It is found that the modification dramatically destroys the native structure of β-lg and results in an obvious increase of the α-helical content, coincident with the accumulation of non-native α-helical intermediates during its folding process. Importantly, the dimeric state of β-lg can still be reached whereas its dimerization rate decreases dramatically, allowing us to characterize the dimerization process using the FRET method based on a stopped-flow apparatus. Our results reveal that the dimerization process occurs before the completely folding of individual monomers, providing direct evidence on the cooperativity of folding and binding processes.