A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/337188
- author
- Bourgeois, D ; Vernede, X ; Adam, V ; Fioravanti, E and Ursby, Thomas LU
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Applied Crystallography
- volume
- 35
- pages
- 319 - 326
- publisher
- International Union of Crystallography
- 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
- 2016-04-01 11:36:06
- date last changed
- 2022-01-26 07:26:21
@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 = {{International Union of Crystallography}},
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}},
doi = {{10.1107/S0021889802003837}},
volume = {{35}},
year = {{2002}},
}