Abstract
Confinement has been demonstrated useful in accelerating the folding process, because of a compact folded protein occupying less volume than an unfolded protein. The scientists have researched on confinement for protein folding, protein diffusion, protein-protein interaction in the recent years. Reverse micelles and mesoporous materials have the common advantages such as high stability, easily tunable size, homogeneous pore structures, and consequently are used to encapsulate molecules for confinement study. In this study, we have explored the confinement effect on biomolecular structure and dynamics by site directed spin label(SDSL) and Cw/pulsed ESR techniques. The molecules we used are tempol, and proteins including Bcl-2-associated X protein(Bax) and T4 lysozyme(T4L). In Part I, we trap the biomolecules in reverse micelles and mesoporous materials to understand the confinement effect. The motions of biomolecules in nanoconfiment are slower than in bulk solution. The results show the confinement effect exists but the structures of biomolecules are distorted slightly. Moreover, we find that the water molecules in the nanochannels stay on amorphous state but not freezing at cryogenic temperature. In Part II, we add trehalose in the water pool of reverse micelles to enhance the confinement effect. The spectra change hardly whether the trehalose is added or not. According to the literature, the trehalose interacts with polar surfactant headgroups. We dope the reverse micelles with spin-labeled lipid(tempo PC, 5-PC) to compose reverse micelles. However, the spectra of the spin-labeled lipid change little with trehalose. Nanoconfinement effects play an important role in protein conformational structure. In this study, we show the conformations of the T4L proteins are somewhat distorted in the mesoporous materials with the pore sizes approximately 8 nm, which is apparently large enough to accommodate the studied proteins. This result is opposed to what we have found for the nano-confined structure of the 26-mer-long polypeptide whose structure was demonstrated to remain unchanged between the bulk solvent and the mesoporous materials. At the current stage, our results point out that the ratio of the pore size and the studied molecular size might be a key to the success for confining a molecule in the nanochannels while leaving its conformation intact. Moreover, it is also possible that the nanoconfinement effects could result in a change of the state in the conformational potential of a protein. Further investigations of the nanoconfinement effect are warranted.