Previous Fluorescence Studies Research Articles

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use 19F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF3-Phe47 labeled M2 peptide and measuring F-CF3 cross peaks in 2D 19F19F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. 1H19F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static 31P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.

High-resolution infrared spectroscopy of supersonically cooled singlet carbenes: Bromomethylene (HCBr) in the CH stretch region.

First high-resolution spectra of cold (∼35K) singlet bromomethylene HCBr in the CH stretching (v1) region from 2770 to 2850cm-1 are reported using near quantum shot-noise limited laser absorption methods in a slit jet supersonic discharge expansion source. Three rovibrational bands are identified at high S/N (20:1-40:1) and rotationally assigned to (i) the CH stretch fundamental (v1) band X̃1,0,0←X̃0,0,0 and (ii) vibrational hot bands [X̃(1,1,0)←X̃(0,1,0) and X̃(1,0,1)←X̃(0,0,1)] arising from vibrationally excited HCBr populated in the discharge with single quanta in either the H-C-Br bend (v2) or C-Br stretch (v3) modes. Precision rotational constants are reported for a total of six states, with an experimentally determined CH stretch vibrational frequency (2799.38cm-1) in good agreement with previous low-resolution fluorescence studies [M. Deselnicu et al., J. Chem. Phys. 124(13), 134302 (2006)]. Detailed analysis of the fundamental v1 band highlights the presence of perturbations in the X̃1,0,0 level, which we tentatively attribute to arise from the nearby triplet state ã(0,0,1) through spin-orbit interaction or the multiple quanta X̃0,2,1 singlet state via c-type Coriolis coupling. Reduced-Doppler resolution (60MHz) in the slit-jet IR spectrometer permits for clear observation of a nuclear spin hyperfine structure, with experimental line shapes well reproduced by nuclear quadrupole/spin-rotation coupling constants from microwave studies [C. Duan et al., J. Mol. Spectrosc. 220(1), 113-121 (2003)]. Finally, the a-type to b-type transition intensity ratio for the fundamental CH stretch band is notably larger than that predicted by using a bond-dipole model, which from high level ab initio quantum calculations [CCSD(T)/PVQZ] can be attributed to vibrationally induced "charge-sloshing" of electron density along the polar C-Br bond.

The spatial biology of transcription and translation in rapidly growing Escherichia coli.

Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0–3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20–30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.

A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus.

The removal of chemically damaged DNA bases such as 3-methyladenine (3-MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge-charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.

Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.

Two-dimensional Fast Surface Imaging Using a Handheld Optical Device: In Vitro and In Vivo Fluorescence Studies

Near-infrared (NIR) optical imaging is a noninvasive and nonionizing modality that is emerging as a diagnostic tool for breast cancer. The handheld optical devices developed to date using the NIR technology are predominantly developed for spectroscopic applications. A novel handheld probe-based optical imaging device has been recently developed toward area imaging and tomography applications. The three-dimensional (3D) tomographic imaging capabilities of the device have been demonstrated from previous fluorescence studies on tissue phantoms. In the current work, fluorescence imaging studies are performed on tissue phantoms, in vitro, and in vivo tissue models to demonstrate the fast two-dimensional (2D) surface imaging capabilities of this flexible handheld-based optical imaging device, toward clinical breast imaging studies. Preliminary experiments were performed using target(s) of varying volume (0.23 and 0.45 cm(3)) and depth (1-2 cm), using indocyanine green as the fluorescence contrast agent in liquid phantom, in vitro, and in vivo tissue models. The feasibility of fast 2D surface imaging ( approximately 5 seconds) over large surface areas of 36 cm(2) was demonstrated from various tissue models. The surface images could differentiate the target(s) from the background, allowing a rough estimate of the target's location before extensive 3D tomographic analysis (future studies).

Connectivity of Photosystem II Is the Physical Basis of Retrapping in Photosynthetic Thermoluminescence

Energy transfer between photosystem II (PSII) centers is known from previous fluorescence studies. We have studied the theoretical consequences of energetic connectivity of PSII centers on photosynthetic thermoluminescence (TL) and predict that connectivity affects the TL Q band. First, connectivity is expected to make the Q band wider and more symmetric than an ideal first-order TL band. Second, the presence of closed PSII centers in an energetically connected group of PSII centers is expected to lower the probability that an exciton originating in a recombination reaction becomes retrapped. The latter effect would shift the Q band toward lower temperature, and the shift would be greater the higher the percentage of closed PSII centers at the beginning of the measurement. These effects can be generalized as second-order effects, as they make the Q band resemble the second-order TL bands obtained from semiconducting solids. We applied the connected-units model of chlorophyll fluorescence to derive equations for quantifying the second-order effects in TL. To test the effect of the initial proportion of closed reaction centers, we measured the Q band with different intensities of the excitation flash and found that the peak position changed by 2.5°C toward higher temperature when the flash intensity was lowered from saturating to 0.39% of saturating. The result shows that energy transfer between reaction centers of PSII forms the physical basis of retrapping in photosynthetic TL. The second-order effects partially explain the deviation of the form of the Q band from ideal first-order TL.

Direct Observation of NS3 Substeps at Single Base Pair Resolution

Helicases are a ubiquitous class of nucleic acid motor proteins. They utilize ATP to catalyze the opening of double-stranded nucleic acids. Previous crystallographic and single molecule fluorescence studies have implied that a single ATP molecule would be used for every base pair (bp) being opened by a helicase. However, this has not been directly observed. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. Previous single molecule optical tweezers studies on NS3 unwinding have revealed an 11-bp periodicity with 3.6 bp substeps on average. By building an optical tweezers instrument with high spatial resolution and low drift, we were able to monitor the discrete unwinding substeps of NS3 on RNA at single base pair resolution. We show that under conditions where ATP binding limits the motor dwell time between successive substeps, single base pair substeps are clearly visible. We present a distribution of these substep sizes and the implication of this result on mechanisms of NS3 catalyzed RNA base pair opening and strand release. The distribution of motor dwell times between substeps gave us a direct measure of the coupling ratio between ATP and base pair opening, and we propose an integrated model to rationalize all these observations.

Characterization Of Ca2+ Photo-release From DM-nitrophen Using Photothermal Beam Deflection

DM-nitrophen is a popular caged calcium compound that allows for a rapid increase in calcium concentration from hundred nanomolar to tens to hundred micromolar level on the microsecond timescale. To fully understand the mechanism of calcium photo-release from DM-nitrophen, we have used photothermal beam deflection technique to investigate time-profiles of volume and enthalpy changes associated with DM-nitrophen photodissociation. Our data show that the photodissociation of calcium loaded DM-nitrophen occurs as a two-step process. The first step takes place within ∼ 10 μs upon photolysis and is associated with a volume decrease of -7 mL mol−1 and enthalpy change of 66 kcal mol−1. On the longer timescale (τ = 200 μs), the second event with a positive volume change of 7 mL mol−1 and enthalpy change of 8 kcal mol−1 was detected. These data are in agreement with the previous fluorescence studies showing that calcium release from DM-nitrophen occurs as a two step process.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Pulsed laser photolysis vacuum UV laser-induced fluorescence kinetic study of the reactions of Cl( 2P 3/2) atoms with ethyl formate, n-propyl formate, and n-butyl formate

Cl( 2P 3/2) atoms were produced by photolysis of Cl 2 at 351 nm in the presence of alkyl formates and monitored by LIF spectroscopy at 134.72 nm. Rate coefficients of k(Cl + C 2H 5OC(O)H) = (9.5 ± 0.3)×10 −12, k(Cl + n-C 3H 7OC(O)H) = (4.5 ± 0.3) × 10 −11 and k(Cl + n-C 4H 9OC(O)H) = (1.2 ± 0.1) × 10 −10 cm 3 molecule −1 s −1 were determined at 295 ± 2 K in 6–7 Torr of Ar diluent. These values are 15–30% lower than reported in a previous pulsed laser photolysis resonance fluorescence study. The discrepancy may reflect complications arising from the fact that the time scales for chlorine atom loss and regeneration were not sufficiently decoupled in the previous study.

Structure, Dynamics, and Branch Migration of a DNA Holliday Junction: A Single-Molecule Fluorescence and Modeling Study

The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.

Metal-Enhanced Fluorescence from Chromium Nanodeposits

Over the last several years, our laboratory has described in detail the favorable effects of fluorophores in close proximity to metallic nanoparticles. Metal nanoparticles comprised of gold, silver, copper, and zinc show enhanced fluorescence intensities and photostabilities for fluorophores positioned within 10 nm of the particles. In this paper we show that, in addition to these metals, chromium nanodeposits can also enhance the fluorescence of close proximity fluorophores, with probes such as fluorescein showing a greater than 8-fold enhancement. However, in contrast to gold, silver, and copper nanoparticles, fluorophores in close-proximity to chromium nanodeposits do not display reduced radiative lifetimes, suggesting that an enhanced electric field component (enhanced excitation rate) is the dominate mode of emission enhancement, similar to recent work by our laboratory for zinc nanoparticles. In addition, we also show the metal-sandwich geometries used in previous metal-enhanced fluorescence studies can give erroneous enhanced fluorescence readings, if care is not taken to normalize the excitation intensity/irradiance.

Detection of Viral Proteins using Human Receptor Functionalized Carbon Nanotubes

ABSTRACTWe present proof-of-concept experiments for developing a highly-sensitive and fast-response miniaturized single-walled carbon nanotube field-effect transistor (SWNT-FET) biosensor for electrically detecting adenovirus using ligand-receptor-protein specificity. SWNTs are mildly oxidized to form carboxylic groups on the surfaces without compromising the electronic integrity of the nanotubes. Then the human coxsackievirus and adenovirus receptor (CAR) is covalently functionalized onto the nanotube surface via diimide-activated amidation process. Upon exposure of the device to adenovirus protein, Ad12 Knob (Knob), specific binding of Knob to CAR decreases the current that flows through the SWNT-FET device. For control experiment, the CAR-SWNT device is exposed to YieF, which is a virus protein that does not bind specifically to CAR, and no current change is observed. The biological activity of the CAR and Knob proteins that are immobilized on SWNTs has been confirmed by previous fluorescence studies [1]. AFM analysis is done to show height increase of a few nanometers at specific spots where the CAR-Knob complex are covalently linked to the nanotube surface. Therefore, our results show that the human receptor protein CAR does immobilize on SWNT surface while fully retains its biological activity. Moreover, the specific binding of CAR to its complementary adenovirus Knob can be electrically detected using individual SWNT-FET devices. These findings suggest that CAR-functionalized SWNT-FETs can ably serve as biosensors for detection of environmental adenoviruses.

Effects of quenching on electronic-resonance-enhanced coherent anti-Stokes Raman scattering of nitric oxide

We investigate the effects of gas-mixture composition on the electronic-resonance-enhanced coherent anti-Stokes Raman scattering (ERE-CARS) signals of nitric oxide (NO). From previous laser-induced fluorescence (LIF) studies, quenching rates are known to change drastically, by factors of 400–800, in mixtures of CO2∕O2∕N2. The observed ERE-CARS signal remains constant to within 30% whereas LIF signals from NO are predicted to decrease by more than two orders of magnitudes in the same environments. This is very significant for using NO ERE-CARS in high-pressure combustion environments where the electronic quenching rate can vary rapidly as a function of both space and time.

Using Single‐Molecule Fluorescence Spectroscopy to Study Electron Transfer

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.

Initial Bubble Collapse Plays a Key Role in the Transition to Elongation in T7 RNA Polymerase

RNA polymerases bind to specific sequences in DNA, melt open duplex DNA around the start site, and start transcription within the initially melted bubble. The initially transcribing complex is relatively unstable, releasing short abortive products. After synthesis of a minimal length of RNA (approximately 10-12 bases in the T7 system), RNA polymerases complete the transition to a processive (highly stable) elongation phase and lose the initial promoter contacts. The current study strongly supports a model for T7 RNA polymerase in which initial bubble collapse from position -4 to position +3 is responsible for initiating RNA displacement in the transition process. More specifically, collapse of the bubble from position -4 to position -1 indirectly and energetically facilitates the direct strand invasion offered by collapse at positions +1 to +3. Parallel work shows that promoter release, another key event occurring during this stage of transcription, begins after translocation to position +8 and is largely complete upon translocation to about position +12. The timing of promoter release agrees with the timing of initial bubble collapse determined by our previous fluorescence studies, suggesting that these two events are closely related.

The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20.

A number of peptides and peptide analogs derived from the membrane proximal region of gp41 ectodomain are found to be effective inhibitors of human immunodeficiency virus type 1 (HIV-1)-mediated fusion events. One of them, T20 (aa 638-673), was found disordered and sparingly soluble in water, but became soluble upon mixing with selected, structured peptides from the amino terminal heptad repeat (HR1) region of gp41 using a simple and sensitive method of reduction in the scattering of T20 suspension. From the results on mapping the locus of interaction with T20 by employing partially overlapping peptides derived from HR1, it was concluded that the LLSGIV segment was a critical docking site for the C-terminal peptide of gp41 in its putative inhibitory action consistent with a previous fluorescence study. It was also found that peptides capable of solubilizing T20 dispersion have a high content of helix, as well as beta-strand, conformation in aqueous solution. Specificity of T20/HR1-derived peptide binding was ascertained by using a scrambled sequence of a T20-active peptide and a plateau in scattering reduction of T20 suspension with variation in the concentration of a T20-active HR1 peptide. Implications on the mechanism of T20 inhibition and the sequence of folding of the gp41 core structure are discussed.

The crystal structure of the sorcin calcium binding domain provides a model of Ca 2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca 2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca 2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca 2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH. The sorcin calcium binding domain (SCBD, residues 33–198) expressed in Escherichia coli was crystallized in the Ca 2+-free form. The structure was solved by molecular replacement and was refined to 2.2 Å with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers. The structure of the SCBD leads to a model that explains the solution properties and describes the Ca 2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca 2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca 2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca 2+-dependent binding to target proteins at or near membranes.

Free-Jet Electronic Spectroscopy of the PO2 Radical

The laser fluorescence excitation spectrum of the jet-cooled PO2 radical has been recorded in the wavelength range 325−284 nm. This work extends previous flash photolysis and laser fluorescence studies of the UV electronic transition in PO2. Resolved emission spectra were recorded for excitation of a number of bands. The previous vibrational analyses of both the absorption and emission spectra were extended. The rotational structure of the bands was investigated and found to be very complicated, defying an analysis. The radiative decays of the excited vibronic levels were found to be nonexponential but could be fitted to a double exponential form. The lifetimes were found to be much shorter than those observed in a discharge-flow system by Hamilton [J. Chem. Phys. 1987, 86, 33]. The inferred dependence of the lifetime upon the degree of rotational excitation and the complicated rotational structure of the bands suggest that there is significant electronic state mixing, as in the isovalent NO2 molecule.