Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane.

Interactions between beta-lactoglobulin (beta-lg) and dimyristoylphosphatidylglycerol (DMPG) bilayers were studied using one- and two-dimensional infrared spectroscopy above (pD 7.4) and below (pD 4.4) the protein's (beta-lg's) isoelectric point (pI=5.2). The aim of the study was threefold: (1) gain a better understanding of beta-lg-phospholipid interaction; (2) provide information relative to the structure of beta-lg as it interacts with membranes; (3) determine whether the conformational modifications of the protein in the presence of lipids are strictly caused by thermal effects or whether they are modulated by the chain-melting phase transition. At pD 7.4, the lipid thermotropism, the acyl-chain order, and the membrane interfacial region were essentially unaffected by the presence of beta-lg, whereas the protein amide I region showed dramatic alterations. The results suggested the predominance of beta-sheets and alpha-helix elements, with a lost of structural integrity. At pD 4.4, beta-lg induced an approximately 2 degrees C downshift of the transition temperature, whereas the conformational order of the lipid chain decreased in the gel phase and increased in the liquid-crystalline phase. The hydration state of the DMPG C==O groups increased in the liquid-crystalline phase. The conformation of beta-lg at pD 4.4 in the presence of DMPG showed similarities with that observed at pD 7.4, but an increase in the alpha-helix content and a reduced thermal stability were noticed. In contrast to the protein alone, beta-lg aggregates in the presence of DMPG at pD 4.4 above 50 degrees C. At both pD values, the charged surface of the membrane seemed to be the main factor for inducing protein conformational changes by altering the intramolecular interactions that stabilize the native structure. However, protein incorporation within the membrane seemed to be involved at pD 4.4. The two-dimensional analysis performed with spectra recorded upon heating showed that spectral intensity changes at pD 4.4 and 7.4 occurred at the same frequencies in the amide I' region. The heat-induced structural changes of beta-lg were not correlated with the conformational modifications of the phospholipids along the phase transition, indicating that the thermal behavior of the protein was not modulated by the lipid chain melting, but rather represented the heat-induced protein rearrangement in the presence of DMPG.