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A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals

Bourgeois, D; Vernede, X; Adam, V; Fioravanti, E and Ursby, Thomas LU (2002) In Journal of Applied Crystallography 35. p.319-326
Abstract
Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown that fluorescence microspectrophotometry might also be used for such purposes if endogenous fluorophores are present in the macromolecule or when exogenous fluorophores are added and either bind to the protein or reside in the solvent channels. An off-line microspectrophotometer that is able to perform low-temperature absorption and fluorescence spectroscopy on crystals mounted in cryo-loops is... (More)
Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown that fluorescence microspectrophotometry might also be used for such purposes if endogenous fluorophores are present in the macromolecule or when exogenous fluorophores are added and either bind to the protein or reside in the solvent channels. An off-line microspectrophotometer that is able to perform low-temperature absorption and fluorescence spectroscopy on crystals mounted in cryo-loops is described. One-shot steady-state emission spectra of outstanding quality were routinely collected from several samples. In some cases, crystals with optical densities that are too low or too high for absorption studies can still be tackled with fluorescence microspectrophotometry. The technique may be used for simple controls such as checking the presence, absence or redox state of a fluorescent substrate/co-factor. Potential applications in the field of kinetic crystallography are numerous. In addition, the possibility to probe key physico-chemical parameters of the crystal, such as temperature, pH or solvent viscosity, could trigger new studies in protein dynamics. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Applied Crystallography
volume
35
pages
319 - 326
publisher
Wiley-Blackwell
external identifiers
  • wos:000175812800005
  • scopus:0036086458
ISSN
1600-5767
DOI
10.1107/S0021889802003837
language
English
LU publication?
yes
id
4c26e797-f46a-43de-8643-ee125edd109b (old id 337188)
date added to LUP
2007-08-02 13:56:49
date last changed
2017-01-01 04:21:35
@article{4c26e797-f46a-43de-8643-ee125edd109b,
  abstract     = {Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown that fluorescence microspectrophotometry might also be used for such purposes if endogenous fluorophores are present in the macromolecule or when exogenous fluorophores are added and either bind to the protein or reside in the solvent channels. An off-line microspectrophotometer that is able to perform low-temperature absorption and fluorescence spectroscopy on crystals mounted in cryo-loops is described. One-shot steady-state emission spectra of outstanding quality were routinely collected from several samples. In some cases, crystals with optical densities that are too low or too high for absorption studies can still be tackled with fluorescence microspectrophotometry. The technique may be used for simple controls such as checking the presence, absence or redox state of a fluorescent substrate/co-factor. Potential applications in the field of kinetic crystallography are numerous. In addition, the possibility to probe key physico-chemical parameters of the crystal, such as temperature, pH or solvent viscosity, could trigger new studies in protein dynamics.},
  author       = {Bourgeois, D and Vernede, X and Adam, V and Fioravanti, E and Ursby, Thomas},
  issn         = {1600-5767},
  language     = {eng},
  pages        = {319--326},
  publisher    = {Wiley-Blackwell},
  series       = {Journal of Applied Crystallography},
  title        = {A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals},
  url          = {http://dx.doi.org/10.1107/S0021889802003837},
  volume       = {35},
  year         = {2002},
}