Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer.
We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar 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 approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-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" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.
Uhrínová
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The Edinburgh Centre for Protein Technology, Joseph Black Chemistry Building, University of Edinburgh, Edinburgh EH9 3JJ, Scotland.
Smith
M H
MH
Jameson
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GB
Uhrín
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D
Sawyer
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Barlow
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PN
D000818:Animals; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D012996:Solutions; D013329:Structure-Activity Relationship
D007782:Lactoglobulins; D010446:Peptide Fragments; D012996:Solutions