[Surface Enhance Infrared Absorption (SEIRAS) have unique properties that enhances signals at vicinity (in an order of several nm's) of a substrate metal. This property is useful to determine solely the surface chemical process distinguished from that occurred in bulk phase. Therefore when a biological sample of interest is confined to such surface, one can selectively monitor a chemical reaction of the target regardless of the complex ensemble biological process in the bulk phase. We present an application of SEIRAS to in-situ investigation for a folding process of membrane protein (bacteriorhodopsin) during cell free expression on the nano-disc artificial lipid bilayer.

Figure 1 A presents a scheme of the experiment with SEIRAS set-up. Single reflection attenuated total reflection (ATR) configuration with gold thin film at reflective surface allows free exchange of the test samples in the bulk phase during measurement of the IR spectra. Surface of SEIRA active gold thin film is modified by an artificial lipid bilayer framed by an amphiphilic scaffold protein (nanodisc). The nanodisc is bound through recombinant affinity tags at the scaffold protein with surface modified Ni-NTA self-assembled monolayer. Mixture of all components for the cell-free expression of bacteriorhodopsin (bR) except translational factor (DNA plasmid) are incubated in the bulk phase at the beginning of the measurement. As the translational factor is added to the mixture, protein synthesis and the folding start to process (Figure 1 B). This process had been monitored by SEIRAS in time resolved manner (Figure 1 C). From these spectra we had succeed to monitor individual reaction step of the translation of bR into the surface confined lipid bilayer.

Figure 1 A) A scheme of the experiment and SEIRAS optical setup; B) In vitro translational process on the gold surface; C) SEIRA spectra observed during the folding of bR into the surface tethered nanodisc monolayer.