Tracking Folding Events During the Cell-Free Expression of Bacteriorhodopsin into Nanodiscs by Seiras

Cell-free expression systems offer a valuable approach to study protein folding in situ. However, studies on such molecular processes over time are difficult to perform because of the disturbing crowded cellular conditions. Applying Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS, Fig. A) can circumvent many of the difficulties. In particular, SEIRAS is exclusively sensitive to processes occurring on the surface. Hence, it is possible to monitor a folding process of a membrane protein by creating a membrane support (here: nanodiscs; Fig. C) on such a surface, in which the target protein can incorporate. Meanwhile, other processes of the cell-free expression system from the bulk will not be visible. We immobilized nanodiscs to a gold surface via His-tag Ni-NTA linkage. By introducing a cell-free transcription/translation protein expression system, the synthesis of bacteriorhodopsin (bR), a seven-helical trans-membrane retinal protein, was initialized (Fig. B). During the reaction infrared spectra were collected in snap-shot mode. Thus, it was possible to monitor events of the translational process in situ. Here, we present secondary structural data as strong evidence for bR folding and incorporation into nanodiscs.