A fluorescein derivative (SAMSA) bound to gold nanoparticles of different diameters is investigated by time-resolved fluorescence at the single molecule level in a wide dynamic range, from nanosecond to second time scale. The significant decrease of both SAMSA excited state lifetime and fluorescence quantum yield observed upon binding to gold nanoparticles can be essentially traced back to an increase of the nonradiative deactivation rate, probably due to energy transfer, that depends on the nanoparticle size. A slow single molecule fluorescence blinking, in the ms time scale, has a marked dependence on the excitation intensity both under single and under two photon excitation. The blinking dynamics is limited by a low probability nonlinear excitation to a high energy state from which a transition to a dark state occurs. The results point out a strong coupling between the vibro-electronic configuration of the dye and the plasmonic features of the metal nanoparticles that provide dye radiationless deactivation channels on a wide dynamic range.

Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.Keywords: Protein immobilization; green fluorescent protein; fluorescence spectroscopy; protein dynamics; silica gels; confocal imagingThe green fluorescent protein (GFP) was discovered in the early 1960s (Shimomura et al. 1962), but only recently it has sparked a lot of interest as a biological tool to monitor complex cellular processes (Chalfie et al. 1994;Cubitt et al. 1995;Heim and Tsien 1996; Chalfie and Kain 1998). The chromophore that confers the typical green color and fluorescent properties to the protein is a p-hydroxybenzylideneimidazole, originated from an internal cyclization at residues Ser65, Tyr66, and Gly67, and 1,2 dehydrogenation of Tyr66 (Cubitt et al. 1995). The three-dimensional structure of the WT GFP and several mutants have been detemined (Ormo et al. 1996;Yang et al. 1996;Brejc et al. 1997;Palm et al. 1997;Wachter et al. 1998;Phillips 1997;Battistutta et al. 2000). The protein shows a ␤-can fold containing an ␣-helix to which the chromophoric moiety is linked. The color is completely but reversibly abolished on unfolding.

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.Keywords: molecular crowding; protein folding; protein immobilization; encapsulation; fluorescence Biological macromolecules are usually studied in diluted solutions. However, the highly crowded cellular environment can have dramatic effects on protein stability, dynamics, and function by modifying solution viscosity, available volume, and solvent and solutes activity (Zimmerman and Minton 1993;Garner and Burg 1994; Minton 2000a Minton ,b, 2001 Ellis 2001a,b). This has recently boosted the interest for investigating biomolecules in artificially crowded and confined environments. Encapsulation in wet nanoporous silica gels, a widely used technique exploited to immobilize and confine protein molecules in a controlled environment (Ellerby et al. 1992;Avnir et al. 1994;Dave et al. 1996;Gill and Ballesteros 2000;Gill 2001;Livage et al. 2001;Mozzarelli and Bettati 2001;Jin and Brennan 2002;Bettati et al. 2004), was recently proved to be a valuable strategy to reproduce in vitro many of the effects of molecular crowding and confinement normally experienced by proteins in vivo (Eggers and Valentine 2001a,b;Klimov et al. 2002). The porous structure of silica gels allows a rapid exchange of solvent and solutes molecules between the gel matrix and the surrounding medium. Excluded volume and altered microviscosity and activity of solvent and solutes inside the gel pores are expected to influence the thermodynamics and kinetics of conformational equilibria. These effects have Reprint requests to: Stefano Bettati, Department of Public Health, Universi...

Fluorescence spectroscopy of a green fluorescent protein mutant at single-molecule resolution has revealed a remarkable oscillatory behavior that can also be driven by applied fields. We show that immediately before unfolding, several periodic oscillations among the chemical substates of the protein chromophore occur. We also show that applied alternating electric or acoustic fields, when tuned to the protein characteristic frequencies, give rise to strong resonance effects.