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Fabio Cannone

Publications and source records attributed to Fabio Cannone.

11 recordsLinked to original sources
PubMed2006

Evidence of discrete substates and unfolding pathways in green fluorescent protein.

We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules' switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.

Fluorescence↗
PubMed2006

Voltage regulation of single green fluorescent protein mutants.

We report the analysis of the fluorescence intensity fluctuations of single proteins of a GFP mutant, GFPmut2, embedded in a polyelectrolyte nanocapsule adsorbed on thin conductive layers. Our results, based on single molecule fluorescence spectroscopy, indicate that the fluorescence blinking dynamics of GFP is strongly dependent on the bulk conductivity of the metal layer substrate, on the distance from the conductive surfaces and on the amplitude of the voltage applied to the poly-electrolyte layers. These findings suggest that fluorescence blinking itself might be employed as a reporter signal in nano-bio-technology applications.

Electrochemistry↗
PubMed2006

Quenching and blinking of fluorescence of a single dye molecule bound to gold nanoparticles.

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.

Biophysical Phenomena↗
PubMed2005

Pre-unfolding resonant oscillations of single green fluorescent protein molecules.

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.

Electricity↗
PubMed2005

Tracking unfolding and refolding of single GFPmut2 molecules.

The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.

Circular Dichroism↗
PubMed2005

Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels.

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.

Circular Dichroism↗
PubMed2004

Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications.

Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.

Fluorescence↗
PubMed2003

Two-photon thermal bleaching of single fluorescent molecules.

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

Fluorescein↗
PubMed2003

Two-photon interactions at single fluorescent molecule level.

Single-molecule spectroscopy and single-molecule detection are emerging areas that have many applications when combined with scanning, imaging, and spectroscopy techniques. We have combined a commercial confocal scanning head, to a Ti:sapphire laser and to an inverted microscope, for the detection of single molecule fluorescence of varies dyes by two-photon excitation. We collected spot images of fluorescent molecules that have been deposited on a substrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interactions that occur with the following fluorescent molecules: Indo-1, Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specific dyes is based upon their wide use in biological and medical applications together with the varying complexity of their chemical structure that increases from Pyrene to Indo-1. Moreover, we report some data about single molecule studies related to denaturation of an enhanced green fluorescent protein, GFPmut2, under one photon excitation regime, that show a very similar trend to that observed for the already mentioned fluorescent molecules.

Fluorescein↗
PubMed2002

Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels.

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.

Anisotropy↗