Structural Changes Accompanying pH-Induced Dissociation of the -Lactoglobulin Dimer
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- 作者：Stanislava Uhrí ; nová ; Mark H. Smith ; Geoffrey B. Jameson ; Dusan Uhrí ; n ; Lindsay Sawyer ; and Paul N. Barlow
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：April 4, 2000
- 年：2000
- 卷：39
- 期：13
- 页码：3565 - 3574
- 全文大小：547K
- 年卷期：v.39,no.13(April 4, 2000)
- ISSN：1520-4995
文摘
We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and tocharacterize the backbone dynamics, of a recombinant version of bovine 
-lactoglobulin (variant A) atpH 2.6, where the protein is a monomer. The structure of this low-pH form of -lactoglobulin is verysimilar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2(crystal) structure, calculated for backbone atoms of residues 6-160, is ~1.3 Å. Differences arise fromthe orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn
-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90),which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" positionat pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of 15N T1s and T2sand 1H-15N heteronuclear NOEs at pH 2.6 and 37 
C. Although the residues of the E-F loop (residues86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well definedby the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Severalresidues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T1 to T2, consistent withconformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3Dstructure of -lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.
-lactoglobulin (variant A) atpH 2.6, where the protein is a monomer. The structure of this low-pH form of -lactoglobulin is verysimilar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2(crystal) structure, calculated for backbone atoms of residues 6-160, is ~1.3 Å. Differences arise fromthe orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90),which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" positionat pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of 15N T1s and T2sand 1H-15N heteronuclear NOEs at pH 2.6 and 37 C. Although the residues of the E-F loop (residues86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well definedby the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Severalresidues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T1 to T2, consistent withconformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3Dstructure of -lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.