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

The stability and dimeric state of𝛽β-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 fluorescenceresonance-energy-βtransfer(FRET)pair of donor(1,5-IAEDANS)and acceptor(5-IAF)dyes to Cys121 of𝛽β-lg monomers to investigate the effect of bulky thiophilic modification on the structure and stability of𝛽β-lg.It is found that the modification dramatically destroys the native structure ofβ-lg and results in an obvious increase of the𝛼-helical content,coincident with the accumulation of non-native𝛼α-helical intermediates during its folding process.Importantly,the dimeric state of𝛽β-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 stoppedflow 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.