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

In English

Författare

Summary, in English

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.

Publiceringsår

2002

Språk

Engelska

Sidor

319-326

Publikation/Tidskrift/Serie

Journal of Applied Crystallography

Volym

35

Dokumenttyp

Artikel i tidskrift

Förlag

International Union of Crystallography

Ämne

  • Natural Sciences
  • Physical Sciences

Status

Published

ISBN/ISSN/Övrigt

  • ISSN: 1600-5767