The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of ∼1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (D L ). Comparison with the expected rate of unfolding in solution (∼2 × 10-3 s-1 at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (N W ) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments (<2 ms), where a membrane-associated native state (N S ) is formed. Unfolding of N S driven by the acidic environment at the membrane surface is proposed to occur via a native-like intermediate lacking Met 80 ligation (M S ), as previously observed during unfolding in solution. The overall unfolding process (N S → D L ) is limited by the rate of disruption of the hydrophobic core in M S . Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence, and circular dichroism showed that D L has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid-denatured equilibrium state (D L ) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (U W ), which has no stable secondary structure.

Cell surface molecules of eukaryotic cells have been considered to be integrated into the membrane bilayer by a transmembrane protein sequence. The Thy-1 antigen of rodent thymocytes and brain was the first eukaryotic membrane molecule for which biochemical data clearly suggested membrane integration via a nonprotein tail. Direct evidence is now presented showing that a glycophospholipid structure is attached to the carboxyl-terminal cysteine residue and that 31 carboxyl-terminal amino acids predicted from the Thy-1 complementary DNA sequence are not present in the mature glycoprotein. These experimental results raise questions concerning signaling across a cell membrane since antibodies to Thy-1 can stimulate T lymphocytes to release lymphokines and undergo cell division.

In aqueous solution, while cytochrome c is a stably folded protein with a tightly packed structure at the secondary and tertiary levels, its heme-free precursor, apocytochrome c, shows all features of a structureless random coil. However, upon interaction with phospholipid vesicles or lysophospholipid micelles, apocytochrome c undergoes a conformational transition from its random coil in solution to an alpha-helical structure on association with lipid. The driving forces of this lipid-induced folding process of apocytochrome c were investigated for the interaction with various phospholipids and lysophospholipids. Binding of apocytochrome c to negatively charged phospholipid vesicles induced a partially folded state with approximately 85% of the alpha-helical structure of cytochrome c in solution. In contrast, in the presence of zwitterionic phospholipid vesicles, apocytochrome c remains a random coil, suggesting that negatively charged phospholipid headgroups play an important role in the mechanism of lipid-induced folding of apocytochrome c. However, negatively charged lysophospholipid micelles induce a higher content of alpha-helical structure than equivalent negatively charged diacylphospholipids in bilayers, reaching 100% of the alpha-helix content of cytochrome c in solution. Furthermore, micelles of lysolipids with the same zwitterionic headgroup of phospholipid bilayer vesicles induce approximately 60% of the alpha-helix content of cytochrome c in solution. On the basis of these results, we propose a mechanism for the folding of apocytochrome c induced by the interaction with lipid, which accounts for both electrostatic and hydrophobic contributions. Electrostatic lipid-protein interactions appear to direct the polypeptide to the micelle or vesicle surface and to induce an early partially folded state on the membrane surface. Hydrophobic interactions between nonpolar residues in the protein and the hydrophobic core of the lipid bilayer stabilize and extend the secondary structure upon membrane insertion.