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 fluorescenceresonance-energy-transfer (FRET) pair of donor (1,5-IAEDANS) and acceptor (5-IAF) dyes to Cys121 of β-lgmonomers 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 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.Bovine β-lactoglobulin (β-lg),a major whey protein abundant in cows milk,has been identified as a member of the lipocalin superfamily of transporter molecules.[1,2] It is predominantly a β-sheet protein composed of a hydrophobic barrel of eight β strands (A-H) and an α-helix at the C-terminal end which protects the oxidation of free thiol group of Cys121 (Fig.1(a)).[3-5] It is known that β-lg can form a dimeric conformation mainly through the hydrogen bonds distributed between the surface of AB loop and the antiparallel β-sheet between theβI strands (Fig.1(a)).