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

• Published in: Chinese physics letters 2011-11, Vol.28 (11), p.118702-1-118702-4
• Main Authors: Pan, Hai (海潘), Xie, Jin-Bing (金兵 谢), Cao, Yi (毅曹), Qin, Meng (猛秦), Wang, Wei (炜王)
• Format: Article
• Language: English
• Subjects: Binding; Dimerization; Dyes; Folding; Fretting; Marking; Monomers; Stability
• ISSN: 0256-307X; 1741-3540
• DOI: 10.1088/0256-307X/28/11/118702

The stability and dimeric state of beta -lactoglobnlin ( beta -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 beta -lg monomers to investigate the effect of bulky thiophilic modification on the structure and stability of beta -lg. It is found that the modification dramatically destroys the native structure of beta -lg and results in an obvious increase of the alpha -helical content, coincident with the accumulation of non-native alpha -helical intermediates during its folding process. Importantly, the dimeric state of beta -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.