DETERMINATION OF THE AMOUNT OF NATIVE STRUCTURAL BACTERIORHODOPSIN INPURPLE MEMBRANE LANGMUIR-BLODGETT-FILMS BY A SPECTROSCOPIC SURFACE DENATURATION QUANTIFYING TECHNIQUE

Purple membrane (PM) shows denaturation when spread over an air/water interface, We established a technique, which we call the spectroscopic surface denaturation quantifying (SSDQ) technique, that uses infrared linear dichroism to determine the amount of native structural bacteri orhodopsin (BR) in PM Langmuir-Blodgett (LB) films, Using the SSDQ tec hnique we found that the conformational change after surface denaturat ion of BR was the same as that caused by ethanol treatment. By extrapo lating the data of the amount of non-denatured BR molecules in PM LB f ilms vs. the area of a single BR molecule on an air/water interface, w e also found that the surface area of a single non-denatured BR molecu le was 11.5 nm(2), which is consistent with that determined by high-re solution electron cryo-microscopy and electron diffraction (EMD). Thes e results demonstrate that the SSDQ technique is effective in quantify ing the amount of native structural BR in PM LB films. The SSDQ techni que is also applicable to other types of protein consisting of alpha-h elical conformation.