Determination of the number of water molecules in the proton pathway of bacteriorhodopsin using neutron diffraction data.

It has been shown that water molecules participate in the proton pathway of bacteriorhodopsin. Large efforts have been made to determine with various biophysical methods the number of water molecules involved. Neutron diffraction H2O/D2O exchange experiments have been often used to reveal the position of water even with low-resolution diffraction data. With this technique, care must be taken with the limitations of the difference Fourier method which are commonly applied to analyze the data. In this paper we compare the results of the difference Fourier method applied to measured diffraction data (not presented here) and models with those from alternative methods introduced here: (1) a computer model calculation procedure to determine a label's scattering length density based on a comparison of intensity differences derived from models and intensity differences from our measurements; (2) a method based on the Parseval formula. Both alternative methods have been evaluated and tested using results of neutron diffraction experiments on purple membranes (Hauss et al. 1994). Our findings indicate that the difference Fourier method applied to low-resolution diffraction data can successfully determine the position of localized water molecules but underestimates their integrated scattering length density in the presence of labels in other positions. Furthermore, we present the results of neutron diffraction experiments on purple membranes performed to determine the number of water molecules in the projected area of the Schiff base at 86%, 75% and 57% relative humidity (r.h.). We found 19 +/- 2 exchangeable protons at 75% r.h., which means at least 8-9 water molecules are indispensable for normal pump function.