Abstract
Protein oligomers are widely found in living organisms and they are also crucial in some neurodegenerative diseases. The aim of this work is to analyze the thermodynamics and stability of protein oligomers employing β-lactoglobulin as the experimental model.β -lactoglobulin is a small, globular and compact protein which, in solution, is in equilibrium with its dimeric form. The association equilibrium can be affected through changes in total protein concentration, temperature and pH. The dimer is the prevalent species at neutral pH. We studied the dissociation process of β-lactoglobulin induced by temperature employing different techniques at pH = 6.7. We performed calorimetric experiments at different values of total protein concentration and we observed that the endotherms of β-lactoglobulin at pH = 6.7 are biphasic and they show a clear dependence with protein concentration. While the transition that appears at higher temperature is insensible to changes in protein concentration the first transition is stabilized when protein concentration is increased. This result allowed proposing a mechanism that involves dissociation of the dimer in the first instance and subsequent denaturing of the monomer. According to the thermodynamic model of a dimer dissociation presented in a previous work (1), it was determined through calorimetric profiles simulations, that only a dimer with negative dimerization enthalpy shows the dependence on protein concentration observed in these experiments. We also performed isothermal titration calorimetric studies and the association parameters of β-lactoglobulin were obtained at pH = 6.7. The dimerization process is exothermic in accordance with the model. Furthermore, temperature induced β-lactoglobulin dissociation at pH = 6.7 was monitored with infrared absorbance spectroscopy and we can state that there are structural changes related to the dissociation of the dimer.(1) J. Phys. Chem. B. 112 (45): 14325-14333, 2008.
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