Joel H. Parks

Fluorescence quenching by the stable free radical di‐t‐butylnitroxide

Bimolecular rate constants (kQ) were measured for the fluorescence quenching of a series of aromatic hydrocarbons by di‐t‐butylnitroxide (DTBN). In methylcyclohexane, measured values of kQ were close to the diffusion limit with only a 6.5‐fold variation through the hydrocarbon series. A study of the viscosity dependence of the quenching reaction, in the Smoluchowski manner, suggests an interaction distance of 4–6 A. A comparison is made of the efficiency of the DTBN and O2 fluorescence quenching reactions. Possible mechanisms are discussed.

FRET measurements of trapped oligonucleotide duplexes

Sensitive methods developed to measure laser-induced fluorescence from trapped ions have been applied to study the intermediate states of the dissociation of oligonucleotide duplexes. Double-stranded oligonucleotide anions were prepared with FRET donor/acceptor pairs attached. Gas phase ions were generated by electrospray ionization and injected into a heated quadrupole ion trap where they were stored and exposed to Nd:YAG laser pulses at 532 nm. The dissociation of the duplexes into single strands was monitored by both fluorescence and mass spectra. The comparison of the degree of dissociation of the duplex observed in the mass spectra to the fluorescence intensity of the donor allows for the determination of the intermediate conformations of the unzipping duplex. The observation of changes in the donor fluorescence in the absence of single strands correlates with an intermediate state characterized by threshold unzipping at the weaker binding end of the duplex. These data are consistent with a model of intermediate state formation as a function of temperature. These studies suggest that FRET methods will be useful to obtain direct measurements, which characterize the dynamics of conformational changes of biomolecules in the gas phase.

Pulsed fluorescence measurements of trapped molecular ions with zero background detection

Sensitive methods have been developed to measure laser-induced fluorescence from trapped ions by reducing the detection of background scattering to zero levels during the laser excitation pulse. The laser beam diameter has been reduced to ∼150 μm to eliminate scattering on trap apertures and the resulting laser-ion interaction is limited to a volume of ∼10−5 cm3 which is ∼0.03–0.15 of the total ion cloud volume depending on experimental conditions. The detection optics collected fluorescence only from within the solid angle defined by laser-ion interaction volume. Rhodamine 640 and Alexa Fluor 350 ions, commonly used as fluorescence resonance energy transfer (FRET) fluorophores, were generated in the gas phase by using electrospray ionization and injected into a radiofrequency Paul trap where they were stored and exposed to Nd:YAG laser pulses at 532 and 355 ran for times up to 10 m. Fluorescence emitted by these ions was investigated for several trap qz values and ion cloud temperatures. Analysis of photon statistics indicated an average of ∼10 photons were incident on the PMT detector per 15 ns pulse for ∼103 trapped ions in the interaction volume. Fluorescence measurements displayed a dependence on trapped ion number which were consistent with calculations of the space charge limited ion density. To investigate the quantitative capability of these fluorescence techniques, the laser-induced fragmentation of trapped Alexa Fluor 350 ions was measured and compared with a rate equation model of the dynamics. Decay of the fluorescence signal as well as the parent ion number compared closely with quantitative predictions of the photofragmentation model.

Simulation of collisional relaxation of trapped ion clouds in the presence of space charge fields

A statistical formulation of ion‐neutral interactions in rf Paul traps is presented which provides a basis for calculating the collisional relaxation of a trapped ion cloud. These calculations describe the ion cloud as a statistical distribution in space and energy and treat collisions by Monte Carlo methods. Ion motion is calculated in the presence of space charge fields in a mean field approximation which allows each ion to be treated independently. This formulation provides an adequate description of the ion cloud dynamics for ion densities ≤108 cm−3. Frequency shifts induced in the ion cloud ensemble by space charge are characterized. Energy relaxation of a C60+ ion cloud initially at a temperature of 5000 K by collisions with a background He gas at 300 K is calculated in the presence of space charge.

Protein fluorescence measurements within electrospray droplets

The conformation of cytochrome c molecules within electrospray droplets is investigated by monitoring the laser induced fluorescence of its single tryptophan residue (Trp-59). By increasing the alcohol concentration of the electrosprayed solutions, protein denaturation is induced, giving rise to significant changes in the intensity of the detected fluorescence. Comparison with analogous denaturation experiments in solution provides information about the relative protein conformations and differences between the bulk-solution and droplet environments. Both electrospray-plume and bulk-solution fluorescence measurements using low methanol concentration solutions indicate the presence of folded protein structures. At high methanol content, fluorescence measurements are consistent with the presence of partly denatured or unfolded conformations. At intermediate methanol content, differences are observed between the extent of denaturation in solution and that within the droplets, suggesting electrosprayed proteins have more compact structures than those detected in bulk measurements using solutions of similar composition. This infers that some fraction of the proteins within the droplets have refolded relative to their bulk-solution conformation. Protein denaturation experiments using the low vapor pressure solvent 1-propanol indicate that differences between the droplet and solution measurements are not due to solvent evaporation effects. It is suggested that different droplet conformations are more likely the result of protein diffusion to the droplet surface and effects of the droplet/air interface. To our knowledge, these are the first reported measurements of protein fluorescence within electrospray droplets

Fluorescence quenching induced by conformational fluctuations in unsolvated polypeptides.

Time-resolved measurements were conducted to relate the fluorescence lifetimes of dye-derivatized polypeptides to local conformational dynamics in trapped, unsolvated peptide ions. This research was performed to better understand the intramolecular interactions leading to the observed increase of fluorescence quenching with temperature and, in particular, how this quenching is related to conformational fluctuations. Dye-derivatized polyproline ions, Dye-[Pro] n -Arg (+)-Trp, are formed by electrospray ionization and trapped in a variable-temperature quadrupole ion trap where they are exposed to a pulsed laser which excites fluorescence. Lifetime data exhibit fluorescence quenching as a result of an interaction between the dye and tryptophan (Trp) side chain. This result is consistent with solution measurements performed for comparison. The lifetime temperature dependence is closely fit over the range 150-463 K by an Arrhenius model of the ensemble averaged quenching rate, k q. Model fits of the measured lifetimes yield a frequency prefactor of approximately 10 (11) s (-1) for k q characteristic of collective motions of the side chains identified in molecular dynamics (MD) simulations. The data fits also yield activation barriers of approximately 0.3 eV, which are comparable to intramolecular electrostatic interactions calculated between the unshielded charge on the Arg residue and the dye. As a result, the quenching rate appears to be determined by the rate of conformational fluctuations and not by the rate of a specific quenching mechanism. The peptide sequence of Dye-Trp-[Pro] n -Arg (+) was also studied and identified a dependence of the quenching rate on the electrostatic field in the vicinity of the dye, Trp pair. Molecular dynamics simulations were performed over the range of experimental measurements to study trajectories relevant to the quenching interaction. The MD simulations indicate that as the temperature is increased, conformational fluctuations in the presence of strong electrostatic fields of the charged Arg (+) residue can result in both (a) an increased number of dye and Trp separations <8 A and (b) increased exothermicity for electron transfer reactions between the dye and Trp. Consequently, the MD simulations are consistent with increased fluorescence quenching with temperature resulting from the occurrence of conformers having specific positions of the dye, Trp, and Arg (+). As a result, the fluorescence lifetime provides a local probe of conformational fluctuations averaged over the ion ensemble.

Fraying and electron autodetachment dynamics of trapped gas phase oligonucleotides.

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double- and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double- and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed.

Cluster experiments in radio frequency Paul traps: Collisional relaxation and dissociation

This paper presents techniques to store, manipulate, and detect cluster ions in an rf Paul trap as an approach to study the physics and chemical physics of clusters. The trap has been designed to be the primary experimental environment in the sense that experimental manipulation and ion detection are performed in situ within the trap. Specific design considerations for the trap and rf electronics relevant to metal cluster experiments are discussed. We present the application of these techniques to measurements of trapped C+60 in order to estimate their limitations for studying metal cluster ions. This paper demonstrates the capability to nondestructively detect small variations in the number of trapped C+60 ions with an ion noise level of ≊100 ions limited by thermal current fluctuations. Trapped ion lifetimes of τion≳30 min are measured following the relaxation of C+60 translational energy to ≊300 K. Measurements of the collisional dissociation of trapped C+60 ions at rates <100 s−1 by He at ∼10−6 Torr h...

Prompt and delayed fluorescence from benzophenone

Abstract Excitation of benzophenone in solution at room temperature using a pulsing nitrogen laser (3371 A, ≈ 6 nsec) leads to prompt fluorescence, phosphorescence, and E-type delayed fluorescence. The first complete spectral distribution of the benzophenone fluorescence is presented.

Collisional quenching kinetics for the HgCl* and HgBr* (B1/2)state

Measurements of the rates of collisional quenching of HgCl* (B 2Σ+1/2) by He, Ne, Ar, Kr, Xe, and N2 at pressures up to 3000 Torr, and HgBr* (B 2Σ+1/2) by He, Ar, Xe, N2, Br2, HBr, CF3Br, and CCl3Br at pressures up to 1000 Torr, are reported in this paper. Steady state measurements are made of HgCl* and HgBr* fluorescence produced by photolyzing HgCl2 and HgBr2, respectively, using Xe2* radiation. A modified Stern–Volmer analysis is used in determining the rates. This analysis accounts for absorption of Xe2* radiation measured for the quenching species Cl2, HCl, CCl4, Br2, HBr, CF3Br, and CCl3Br. The measured vacuum UV cross sections for these species are included.