First use of the UV Super-ACO free-electron laser: Fluorescence decays and rotational dynamics of the NADH coenzyme

Abstract

Significant improvements in the performances of the Super-ACO storage ring free-electron laser (FEL) at 800 MeV have been obtained recently: enhancement of the output power in the ultraviolet, laser duration of 10 h for the same injection of positrons, long-term stability with a micropulse of 60 ps FWHM. A first series of experiments using this FEL has then been successfully performed. Taking advantage of the time structure, the polarization and the high power of the source at 350 nm, the polarized fluorescence decays of the reduced nicotinamide adenine dinucleotide coenzyme (NADH) were studied in aqueous solution, using the single-photon counting (SPC) technique. The experimental setup is described, including the Super-ACO FEL characteristics and diagnostics. The FEL working point has been first optimized by monitoring the SPC apparatus function. A complete fluorescence experiment required about 30 min of data acquisition, during which the best integrated instrumental response had a FWHM of 110 ps. Measurements performed in such a way lead to the unambiguous separation of two close lifetime components of 0.28 and 0.62 ns in the fluorescence decays of NADH at 20 °C, in good agreement with previous works. The thermodynamic parameters obtained from temperature studies show that the NADH fluorescence heterogeneity is consistent with the ground-state folding equilibrium of the coenzyme, as characterized by many other spectroscopic techniques. From the fluorescence anisotropy decays, an apparent hydrodynamic radius of about 6 Å is determined, while on the other hand, a large initial depolarization of the fluorescence indicates a fast independent motion of the nicotinamide ring. The quality of the collected data fully meets the requirements for the study of more complex systems such as fluorescent compounds bound to proteins or membranes. Thus, the feasibility of use of a storage ring UV FEL for this type of time-resolved experiments on the subnanosecond time scale has been demonstrated.

Conformational Dependence of Intracellular NADH on Metabolic State Revealed by Associated Fluorescence Anisotropy*♦

“…We measure the total fluorescence decay to be bi-exponential with lifetimes (and amplitudes) 350 ps (0.77) and 760 ps (0.23) giving an average lifetime of 444 ps. The two lifetime components are comparable with those reported in literature and are believed to arise from the extended and folded populations respectively (12,21). The fluorescence anisotropy decay was found to be mono-exponential, with a rotational diffusion time of 290 ps and an initial anisotropy of 0.38 (Fig.…”

“…This relation approximates the hydrodynamic volume (molecule ϩ hydrodynamic shell) of the molecule to be spherical, which is a reasonable approximation for NADH (21). We use this relation later to differentiate between free and bound NADH as well as to estimate the viscosity of the intracellular environment.…”

Le laser à électrons libres UV de super-ACO : source et applications

“…Après une expérience à un photon LEL de déclin de fluorescence polarisée en biologie [4], les applications se sont portées sur des expériences pompe/sonde à deux couleurs combinant le LEL et le Rayonnement Synchrotron (RS). Grâce à la synchronisation naturelle entre les deux sources, et à leur accordabilité, on peut effectuer des expériences résolues en temps et/ou en fréquence adaptées à l'études d'états excités et plus généralement de systèmes hors d'équilibre [5].…”

Lasers UV à électrons libres

“…Des résultats originaux ont pu être obtenus, concernant l'équilibre thermodynamique entre les différents états conformationnels, et le volume hydrodynamique de l'état replié [19]. Les LELs sur anneaux de stockage sont particulièrement bien adaptés aux expériences pompe-sonde avec le rayonnement synchrotron, naturellement synchronisé avec le LEL.…”