Title: Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements
Authors: Royant, Antoine
Carpentier, Philippe
Ohana, Jeremy
McGeehan, John
Paetzold, Bernhard
Noirclerc-Savoye, Marjolaine
Vernede, Xavier
Adam, Virgile
Bourgeois, Dominique # ×
Issue Date: Dec-2007
Publisher: Blackwell Publishing on behalf of the International Union of Crystallography
Series Title: Journal of Applied Crystallography vol:40 pages:1105-1112
Abstract: Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV-visible absorbance or fluorescence mode have evolved over the past few decades to become the instruments of choice to perform such tests. Described here are recent improvements to the microspectrophotometer of the so-called Cryobench laboratory located at the European Synchrotron Radiation Facility, Grenoble, France. Optical and mechanical properties have been enhanced so as to record better spectra on smaller samples. A device has been implemented to measure the signal decay of fluorescent samples, either in the crystalline or in the solution state. Recording of the fluorescence lifetime in addition to the steady-state fluorescence emission spectrum allows precise monitoring of the fluorescent sample under study. The device consists of an adaptation of a commercially available time-correlated single-photon-counting (TCSPC) system. A method to record and analyze series of TCSPC histograms, e. g. collected as a function of temperature, is described. To validate the instruments, fluorescence lifetimes of fluorescent small molecules or proteins in the crystalline or solution state, at room and cryo temperatures, have been measured. Lifetimes of a number of fluorescent proteins of the GFP family were generally found to be shorter in crystals than in solution, and slightly longer at cryo temperatures than at ambient temperature. The possibility of performing fluorescence lifetime measurements on crystals at synchrotron facilities widens the variety of spectroscopic techniques complementing X-ray diffraction on macromolecular crystallography beamlines.
ISSN: 0021-8898
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Imaging and Photonics
× corresponding author
# (joint) last author

Files in This Item:
File Description Status SizeFormat
2002_J.App.Crys..pdfJAppCrys_2007 Published 578KbAdobe PDFView/Open


All items in Lirias are protected by copyright, with all rights reserved.

© Web of science