Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3.

Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.

Thermodynamics and mechanism of protonated cysteine decomposition: a guided ion beam and computational study.

A quantitative molecular description of the decomposition of protonated cysteine, H(+)Cys, is provided by studying the kinetic energy dependence of threshold collision-induced dissociation (CID) with Xe using a guided ion beam tandem mass spectrometer (GIBMS). Primary dissociation channels are deamidation (yielding both NH3 loss and NH4(+) formation) and (H2O + CO) loss reactions, followed by an additional six subsequent decompositions. Analysis of the kinetic energy-dependent CID cross sections provides the 0 K barriers for six different reactions after accounting for unimolecular decay rates, internal energy of reactant ions, multiple ion-molecule collisions, and competition among the decay channels. To identify the mechanisms associated with these reactions, quantum chemical calculations performed at the B3LYP/6-311 + G(d,p) level were used to locate the transition states (TSs) and intermediates for these processes. Single point energies of the reactants, products, and key optimized TSs and intermediates are calculated at B3LYP, B3P86, and MP2(full) levels using a 6-311 + G(2d,2p) basis set. The computational characterization of the elementary steps of these reactions, including the structures of the final products, is validated by quantitative agreement with the experimental energetics. In agreement with previous work, deamidation is facilitated by anchimeric assistance of the thio group, which also leads to an interesting rearrangement of the intact amino acid identified computationally.

Photophysical and dynamical properties of doubly linked Cy3-DNA constructs.

Photophysical measurements are reported for Cy3-DNA constructs in which both Cy3 nitrogen atoms are attached to the DNA backbone by short linkers. While this linking was thought to rigidify the orientation of the dye and hinder cis-isomerization, the relatively low fluorescence quantum yield and the presence of a short component in the time-resolved fluorescence decay of the dye indicated that cis-isomerization remained possible. Fluorescence correlation spectroscopy and transient absorption experiments showed that photoisomerization occurred with high efficiency. Molecular dynamics simulations of the trans dye system indicated the presence of stacked and unstacked states, and free energy simulations showed that the barriers for stacking/unstacking were low. In addition, simulations showed that the ground cis state was feasible without DNA distortions. Based on these observations, a model is put forward in which the doubly linked dye can photoisomerize in the unstacked state.

Photophysical processes in single molecule organic fluorescent probes.

The use of organic fluorescent probes in biochemical and biophysical applications of single molecule spectroscopy and fluorescence microscopy techniques continues to increase. As single molecule measurements become more quantitative and new developments in super-resolution imaging allow researchers to image biological materials with unprecedented resolution, it is becoming increasingly important to understand how the properties of the probes influence the signals measured in these experiments. In this review, we focus on the photochemical and photophysical processes of organic fluorophores that affect the properties of fluorescence emission. This includes photobleaching, quenching, and the formation of non-emissive (dark) states that result in fluorescence blinking in a variety of timescales. These processes, if overlooked, can result in an erroneous interpretation of the data. Understanding their physical origins, on the other hand, allows researchers to design experiments and interpret results so that the maximum amount of information about the system of interest can be extracted from fluorescence signals.

Photobleaching and blinking of TAMRA induced by Mn(2+).

Blinking and bleaching: Coordination of Mn(2+) to DNA induces intersystem crossing, causing fluctuations in the fluorescence intensity and accelerated photobleaching.

pH-dependent spectral properties of para-aminobenzoic acid and its derivatives.

The local environment dictates the structural and functional properties of many important chemical and biological systems. The impact of pH on the photophysical properties of a series of para-aminobenzoic acids is examined using a combination of experimental spectroscopy and quantum chemical calculations. Following photoexcitation, PABA derivatives may undergo an intramolecular charge transfer (ICT) resulting in the formation of a zwitterionic species. The thermodynamics of the excited state reaction and temperature-dependence of the radiative emission processes are evaluated through variable temperature fluorescence spectroscopy carried out in a range of aqueous buffers. Quantum chemical calculations are used to analyze structural changes with modifications at the amine position and different protonation states. The ICT is only observed in the tertiary amine, which calculations show has more sp(2) character than the primary or secondary amines. Thermodynamic analysis indicates the ICT reaction is driven by entropy.

An experimental and theoretical study of alkali metal cation interactions with cysteine.

The interactions of alkali metal cations (M(+) = Li(+), Na(+), K(+), Rb(+)) with the amino acid cysteine (Cys) are examined in detail. Experimentally, bond energies are determined using threshold collision-induced dissociation of the M(+)(Cys) complexes with xenon in a guided ion beam mass spectrometer. Analyses of the energy dependent cross sections provide 0 K bond energies of 2.65 +/- 0.12, 1.83 +/- 0.05, 1.25 +/- 0.03, and 1.06 +/- 0.03 eV for complexes of Cys with Li(+), Na(+), K(+), and Rb(+), respectively. All bond energy determinations include consideration of unimolecular decay rates, internal energy of reactant ions, and multiple ion-molecule collisions. Ab initio calculations at the MP2(full)/6-311+G(2d,2p), B3LYP/6-311+G(2d,2p), and B3P86/6-311+G(2d,2p) levels with geometries and zero-point energies calculated at the B3LYP/6-311G(d,p) level for the lighter metals show good agreement with the experimental bond energies. For Rb(+)(Cys), similar calculations using the HW* basis set and ECP underestimate the experimental bond energies, whereas the Def2TZVP basis set yields results in good agreement. Ground state conformers are tridentate for Li(+) and Na(+), and subtle changes in the Cys side-chain orientation are found to cause noticeable changes in the alkali metal binding energy. For K(+) and Rb(+), tridentate and carboxylic acid bound (both charge-solvated and zwitterionic) structures are nearly isoenergetic, with different levels of theory predicting different ground conformers. The combination of this series of experiments and calculations allows the influence of the sulfur functional group of Cys on the overall binding strength to be explored. Comparison to previous results for serine elucidates the influence of sulfur for oxygen substitution.

Structural and spectroscopic studies of the photophysical properties of benzophenone derivatives.

The effect a solvent has on the photophysical properties of a series of benzophenone derivatives, all FDA approved for use in sunscreens, is examined. Experimentally significant differences in the solvatochromic behavior are found to be dependent upon the substituents on the parent benzophenone molecule. The spectral trends do not appear to originate from only changes in the solvent polarity but indicate that specific solvent-solute interactions influence the absorbance energies of some benzophenones. Computational investigations examine the structure and electronic excitation energies of the molecules. Specific interactions of the solvent and solute are modeled to evaluate structural changes that result from solvent-solute complexation and the impact of the changes upon absorbance properties. The viability of an intramolecular excited state proton transfer is theoretically evaluated. The combination of experimental and computational analysis provides a more complete understanding of the molecular level origin of the unique photophysical properties of this class of UV absorbers.