Microsecond exchange of internal water molecules in bacteriorhodopsin

The proton-conducting pathway of bacteriorhodopsin (BR) contains at least n ine internal water molecules that are thought to be key players in the prot on translocation mechanism. Here, we report the results of a multinuclear ( H-1, H-2, O-17) magnetic relaxation dispersion (MRD) study with the primary goal of determining the rate of exchange of these internal water molecules with bulk water. This rate is of interest in current attempts to elucidate the molecular details of the proton translocation mechanism. The relevance of water exchange kinetics is underscored by recent crystallographic findi ngs of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is beli eved to be governed by conformational fluctuations in the protein and can t herefore provide information about the thermal accessibility of functionall y important conformational substates. The present H-2 and O-17 MRD data sho w that at least seven water molecules, or more if they are orientationally disordered, in BR have residence times (inverse exchange rate constant) in the range 0.1-10 mus at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1-0.5 mus. These results sh ow that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short con pared to the rate-limiting step in the photo cycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of det ergent-solubilized BR was determined to 35 ns at 300 K, consistent with a m onomeric protein in complex with about 150 detergent molecules. The solubil ized protein was found to be stable in the dark for at least eight months a t 277 K. (C) 2001 Academic Press.