Analysis of refolding process of proteins on lipid planar membranes

抄録

Introduction: Recently, membrane proteins and some of cellular proteins can be (re)folded on the model biomembranes such as liposomes (a closed phospholipid bilayer system), although the details for the molecular mechanism are still now unclear. In this study, we then applied a single molecular tracking method to the model globular protein, carbonic anhydrase from bovine (CAB) to investigate its refolding process on lipid planar membranes.

Methods: We used two kinds of phosopholipids such as 1,2-dipalmitoyl phosphatidylcholine (DPPC, gel phase) and 1,2-dioleoyl phosphatidylcholine (DOPC, liquid crystallin phase) to prepare the lipid planar membranes. GuHCl-denatured CAB was injected on the lipid membranes to start the refolding reaction process. For a single molecular tracking experiment, CAB was modified with fluorescence probe DAC.

Results: CAB took conformation of native (N) and unfolded state (U), depending on the GuHCl concentration. A lateral diffusion coefficient (D) of CAB on lipid membranes was then determined. The D value depended on the conformation of CAB. Then, the refolding process was monitoring based on D value. In the beginning of refolding, CAB molecules indicated the low D value due to the denaturede state. Some molecules came to indicate the incremental change of D value. This result implies that some of CAB molecules immediately convert from U->I->N state (I intermediate state). Furthere experiment demonstrated that the lateral diffusivity of I state consisted of (i) slaved-diffusion and (ii) adsorption-desorption steps. These steps strongly depended on the gel / liquid-crystalline phase of DOPC and DPPC membranes. By repeating these steps, the refolding reaction process of CAB was advanced, as shown in Fig.1. These results would be helpful to understand the lipid membrane-assisted refolding of other proteins.