Pressure-Induced Subunit Dissociation and Unfolding of Dimeric β-Lactoglobulin

Abstract

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.

Acidic pH Modulates the Interaction between Human Granulocyte-Macrophage Colony-stimulating Factor and Glycosaminoglycans

“…Once dissociated by dilution to 5 g/ml (ϳ0.35 M) in neutral buffer, these GM-CSF oligomers might not reassociate by pH acidification, either because of the addition of heparin or because the oligomerization process does not reach equilibrium in the time frame of the experiment. Phenomena related to the lack of fast equilibrium for oligomerization have been previously described for protein dimers and other oligomers (41,42). It is interesting to note that incubation of the acidified sample (open circle far left in Fig.…”

Effects of Hydrostatic High Pressure on the Structure and Antibacterial Activity of Recombinant Human Lactoferrin from Transgenic Rice

“…28,29) If -lactoglobulin is in an unfolding environment, under high pressure, the free SH group of Cys121 appears to be able to interact irreversibly with disulfide bonds. 30) However, this is not the case of lactoferrin, which does not have a free thiol group in the molecule. 31) The existence of a single peak in DSC thermograms indicates the same thermosensitivity and simultaneous denaturation of the two lobes of lactoferrin.…”

A Direct Method for the Correction of Pressure-Induced Scrambling of Polarized Fluorescence Intensities

“…Values for E and G (the excitation and grating factors, respectively) were determined (for simplicity), using the sample of interest according to Eq. [5], with p ϭ 1 bar. However, a more rigorous approach for determination of these factors requires measurement of an appropriate dye dissolved in an isotropic solvent (e.g., hexane or methanol), using the more conventional 10 ϫ 10-mm square quartz cuvette under the same optical (excitation and emission) conditions as those employed for the high-pressure studies.…”

“…[3], [4], and [5]. After input of E and G, and for a given pressure the polarized emission intensities (i VV , i VH , i HH , i HV ) for the sample of interest, the values of X( p) and Y( p) and then ͗r͘ corr can be retrieved automatically and directly.…”

“…Due to the noncompressibility of the aqueous solvent, applied pressure effects on the observed fluorescence emission anisotropy reflect exclusive alteration in the hydrodynamic volume of the system under investigation. Hence, protein conformations (1-4), dissociation and association of oligomeric proteins (1,5,6), and altered lipid membrane structure (1,(7)(8)(9) and/or dynamics (10 -12) can be readily studied at concentration levels of noninfinite dilution. In addition, the technique can provide information regarding local flexibility or overall rotational dynamics of a system, depending on the nature of the fluorophore studied (1,2,4,13).…”