期刊
BIOPHYSICAL JOURNAL
卷 103, 期 2, 页码 303-312出版社
CELL PRESS
DOI: 10.1016/j.bpj.2012.05.041
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类别
资金
- Riddet Institute
- University of Auckland
- CR Roper Bequest
- New Zealand Royal Society [UOC1013]
- U.S. Army Research Laboratory
- U.S. Army Research Office [W911NF-11-1-0481]
The oligomerization of beta-lactoglobulin (beta Lg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of beta Lg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25 degrees C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for beta Lg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K-D(2-1) fell close to or within the protein concentration range of similar to 5 to similar to 45 mu M, and at similar to 45 mu M the dimer predominated. No species larger than the dimer could be detected. The K-D(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI similar to 5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding beta Lg play a key role in the thermodynamics and kinetics of dimer association/dissociation.
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