EFFECT OF LIPIDS ON THE THERMAL-STABILITY AND CONFORMATIONAL-CHANGES OF PROTEINS - RIBONUCLEASE-A AND CYTOCHROME-C

Lipids have been increasingly used as carriers for delivery of protein s and peptides. In this study, thermal stability and conformational pr operties of two basic proteins, ribonuclease A (RNase A) and cytochrom e c (cyt, c), incorporated in lipid membranes made with dipalmitoylpho sphatidylglycerol (DPPG), a negatively charged lipid, was studied by d ifferential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. DSC studies showed that when incorporated in DPP G at concentration < 4 mol%, these two proteins were stabilized by DPP G binding, and > 4 mol%, a destabilizing effect was observed. Such a d ecrease in thermal stability of cyt. c or RNase A suggested a strong i ntermolecular protein-protein interaction, because of the relatively l ow lipid to protein ratio. When cyt. c bound to membranes made of a mi xture of DPPG and DPPC (dipalmitoylphosphatidylcholine), the extent of structural perturbation depended on the surface density of the negati vely charged lipid head groups; perturbation became smaller with incre asing acidic phospholipid in the membrane. FT-IR studies showed a shif t of the major amide I component band from 1653 to 1649 cm(-1) for cyt . c and from 1639 to 1633 cm(-1) for RNase A after incorporation into DPPG membranes. However, from the quantitative determination of Fourie r self-deconvoluted spectra, only slight perturbation of the secondary structure was observed after DPPG binding. These reductions, along wi th the shifts to lower frequencies of the main component bands, sugges ted that some rearrangement within the alpha helices/beta sheets and/o r the loosening of the protein tertiary structure existed, resulting i n a stronger hydrogen bonding accessibility after their binding to DPP G membranes. (C) 1998 Elsevier Science B.V.