Prolonged lifetime of the M intermediate in D96N-mutant bacteriorhodopsin films enhanced by diaza-15-crown-5

Abstract

In order to get even longer M intermediate lifetime for the purpose of expanding the use of bacteriorhodopsin (BR) materials in optical applications, the combination of genetic engineering and the use of chemical additives was attempted. Different compositions of BR (wild type and D96N mutant)–PVA films with diaza-15-crown-5 (1,4,10-trioxa-7,13-diazacyclopentadecane) additive were prepared. Spectral and kinetic measurements were carried out at room temperature. It was observed that the additive contents that could be used in these films were limited. The highest BR∶diaza-15-crown-5 molecular ratios were 1∶250 and 1∶150 for BR_WT and BR_D96N films, respectively. Further additions resulted in permanent damage to the purple membrane and a loss of photochromism. The kinetic curves of the M state decay for each film were fit, in a least-squares fashion, by a three-exponential function to obtain sufficiently small residuals. The fitting results indicate that for both types of BR molecules, the decay of the M state was slowed down gradually with increasing additive content. At a BR_D96N∶diaza-15-crown-5 molecular ratio of 1∶150, the BR film had the longest lifetime and its photochromism could be observed for about one and a half hours. This indicates that the combination of genetic engineering and chemical additives is more efficient than using either of these two methods. The main reason for this significant prolongation may originate from the strong basic and H⁺ complexing properties of diaza-15-crown-5 and the change of the membrane surface charge caused by this additive.