期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 356, 期 2, 页码 483-496出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2005.11.038
关键词
beta-lactoglobulin; Tanford transition; NMR; loop dynamics; pH-dependent conformational change
The Tanford transition is a conformational change of bovine beta-lactoglobulin (beta LG) occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the beta-barrel of the beta LG molecule below pH 7 and flipping to open the cavity above pH 7. To clarify the dynamics of this conformational change, NMR measurements were made at neutral pH. Since severe signal broadening due to monomer-dimer equilibrium prevented NMR measurements of wildtype beta LG at neutral pH, we searched for optimal sample conditions, finding that a disulfide bond-linked dimer of the mutant A34C gives an HSQC spectrum without signal broadening. The HSQC and CD spectra indicated that in overall conformation A34C is similar to wild-type beta LG, suggesting that the A34C dimer is a good model with which to study the structure and dynamics of the wild-type at neutral pH. The pH-dependent HSQC signal changes and Lipari-Szabo type relaxation analyses of the A34C dimer revealed that the conformational change to the EF-loop occurs above pH 7. We observed two types of motions in the EF-loop region; relatively fast (micro- to milliseconds) and slow (milliseconds or slower) conformational exchanges of the residues located in the hinge and top of the EF-loop regions, respectively. Furthermore, the GH-loop adjacent to the EF-loop exhibited conformational change at a pH slightly lower than that at which the EF-loop motions occurred. From these observations, we propose a three-step mechanism of conformational change in the EF-loop leading to the Tanford transition, in which the GH-loop conformational change, the cleavage of the hydrogen bonds at the hinge, and the flip of the EF-loop occur sequentially. (c) 2005 Elsevier Ltd. All rights reserved.
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