Absorption spectroscopy of three-dimensional bacteriorhodopsin crystals at cryogenic temperatures: effects of altered hydration.
A comparative study of absorption spectroscopy at 100 K has been performed on three-dimensional crystals of bacteriorhodopsin extracted from a lipidic cubic phase and on native purple membrane. A modified microspectrophotometer has been designed which yields absorption data with a high signal-to-noise ratio and remarkable reproducibility. Excellent agreement of the absorption spectra of the three-dimensional crystals and the purple membrane is observed provided that a rigorous crystal-handling procedure is followed. This result supports the equivalence of the protein structure in both the cubic phase crystals and the native purple membrane. On the other hand, it is shown that dramatic deviations of the crystal spectrum can be induced by minor changes in the extraction method. Exposure to air at room temperature can lead within a short time to an irreversible dehydration manifested by a distinct species with an absorption maximum at 500 nm. Exposure of the crystals to a buffer with lower ionic strength than the crystallization solution produces a different spectral form with an absorption maximum at 477 nm, which was assigned to a distorted protein conformation induced by osmotic stress. The extreme sensitivity of these crystals to experimental conditions is relevant for X-ray structural studies, in particular as different experimental treatments are implemented to trap the intermediates of the protein's photocycle.
Portuondo-Campa
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E
Ecole Polytechnique Fédérale de Lausanne, Laboratory of Ultrafast Spectroscopy, ISIC, FSB-BSP, CH-1015 Lausanne, Switzerland.
Schenkl
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S
Dolder
M
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Chergui
M
M
Landau
E M
EM
Haacke
S
S
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002021:Buffers; D003080:Cold Temperature; D003460:Crystallization; D003461:Crystallography; D019617:Halobacterium salinarum / Q000737:chemistry; D018717:Purple Membrane / Q000737:chemistry; D015203:Reproducibility of Results; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods; D014867:Water / Q000737:chemistry*
D002021:Buffers; D014867:Water; D001436:Bacteriorhodopsins