期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 381, 期 1, 页码 89-101出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.05.018
关键词
allostery; HDX-MS; MALDI-ToF MS; biotin repressor
资金
- NCRR NIH HHS [S10 RR019341, S10RR19341] Funding Source: Medline
- NIGMS NIH HHS [GM46511, R37 GM021248, R01 GM046511-17, R01 GM046511, GM21248, R01 GM021248] Funding Source: Medline
The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5'-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding. (C) 2008 Elsevier Ltd. All rights reserved.
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