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Microsecond Exchange of Internal Water Molecules in Bacteriorhodopsin

Gottschalk, Michael LU ; Dencher, Norbert A and Halle, Bertil LU (2001) In Journal of Molecular Biology 311(3). p.605-621
Abstract
The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) 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 findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is... (More)
The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) 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 findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is believed to be governed by conformational fluctuations in the protein and can therefore provide information about the thermal accessibility of functionally important conformational substates. The present 2H and 17O MRD data show 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 μs at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1–0.5 μs. These results show that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short compared to the rate-limiting step in the photocycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of detergent-solubilized BR was determined to 35 ns at 300 K, consistent with a monomeric protein in complex with about 150 detergent molecules. The solubilized protein was found to be stable in the dark for at least eight months at 277 K. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Molecular Biology
volume
311
issue
3
pages
605 - 621
publisher
Elsevier
external identifiers
  • scopus:0035902770
ISSN
1089-8638
DOI
10.1006/jmbi.2001.4895
language
English
LU publication?
yes
id
fb9360d9-e072-4c5e-bbef-1d4fcfaa0c84
date added to LUP
2017-02-23 11:24:07
date last changed
2018-01-07 11:52:23
@article{fb9360d9-e072-4c5e-bbef-1d4fcfaa0c84,
  abstract     = {The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) 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 findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is believed to be governed by conformational fluctuations in the protein and can therefore provide information about the thermal accessibility of functionally important conformational substates. The present 2H and 17O MRD data show 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 μs at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1–0.5 μs. These results show that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short compared to the rate-limiting step in the photocycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of detergent-solubilized BR was determined to 35 ns at 300 K, consistent with a monomeric protein in complex with about 150 detergent molecules. The solubilized protein was found to be stable in the dark for at least eight months at 277 K.},
  author       = {Gottschalk, Michael and Dencher, Norbert A and Halle, Bertil},
  issn         = {1089-8638},
  language     = {eng},
  number       = {3},
  pages        = {605--621},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Biology},
  title        = {Microsecond Exchange of Internal Water Molecules in Bacteriorhodopsin},
  url          = {http://dx.doi.org/10.1006/jmbi.2001.4895},
  volume       = {311},
  year         = {2001},
}