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 ¯uorescence microspectrophotometry
might also be used for such purposes if endogenous ¯uorophores
are present in the macromolecule or when exogenous ¯uorophores 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 ¯uorescence 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
¯uorescence microspectrophotometry. The technique may be used for simple
controls such as checking the presence, absence or redox state of a ¯uorescent
substrate/co-factor. Potential applications in the ®eld 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.