Infrared Monitoring of Interlayer Water in Stacks of Purple Membranes†

Abstract

The thermodynamic behavior of films of hydrated purple membranes from Halobacterium salinarum and the water confined in it was studied by Fourier transform infrared spectroscopy in the 180-280 K range. Unlike bulk water, water in the thin layers sandwiched between the biological membranes does not freeze at 273 K but will be supercooled to approximately 256 K. The melting point is unaffected, leading to hysteresis between 250 and 273 K. In its heating branch, a gradually increasing light-scattering by ice is observed with rate-limiting kinetics of tens of minutes. Infrared (IR) spectra decomposition provided extinction coefficients for the confined water vibrational bands and their changes upon freezing. Because of the hysteresis, at any given temperature in the 255-270 K range, the interbilayer water could be either liquid or frozen, depending on thermal history. We find that this difference affects the dynamics of the bacteriorhodopsin photocycle in the hysteresis range: the decay of the M and N states and the redistribution between them are different depending on whether or not the water was initially precooled to below the freezing point. However, freezing of interbilayer water does block the M to N transition. Unlike the water, the purple membrane lipids do not undergo any IR-detectable phase transition in the 180-280 K range.