摘要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.
Abstract：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.
PAN Hai;XIE Jin-Bing;CAO Yi**;QIN Meng**;WANG Wei
. Effects of Labeling Thiophilic FRET Dyes on the Stability and Dimerization Process of β-Lactoglobulin[J]. 中国物理快报, 2011, 28(11): 118702-118702.
PAN Hai, XIE Jin-Bing, CAO Yi**, QIN Meng**, WANG Wei
. Effects of Labeling Thiophilic FRET Dyes on the Stability and Dimerization Process of β-Lactoglobulin. Chin. Phys. Lett., 2011, 28(11): 118702-118702.
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