摘要
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.
作者
潘海
谢金兵
曹毅
秦猛
王炜
PAN Hai;XIE Jin-Bing;CAO Yi;QIN Meng;WANG Wei(National Laboratory of Solid State Microstructure and Department of Physics,Nanjing University,Nanjing 210093)
基金
by the National Natural Science Foundation of China under Grant Nos 10904064,10834002 and 11074115
the Natural Science Foundation of Jiangsu Province under Grant No BK2009008.