Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence

2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic decreases in quantum yield and emission decay lifetime that vary sensitively with base sequence, temperature, and helix conformation but that are accompanied by only small changes in emission wavelength. However, the molecular interactions that give rise to these spectroscopic changes have not been established. To develop a molecular model for interpreting the fluorescence measurements, we have investigated the effects of environmental polarity, hydrogen bonding, and the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and intensity decay kinetics of 2AP in simple model systems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investigated in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide (DMF). The effects of the purine and pyrimidine bases were studied by titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the lifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static quenching is saturable and has half-maximal effect at approximately 20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence lifetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effects of polarity do not coincide with the observed emission wavelength-independent quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence quenching of 2AP in DNA arises from base stacking and collisions with neighboring bases only but is insensitive to base-pairing or other hydrogen bonding interactions. These results implicate both structural and dynamic properties of DNA in quenching of 2AP and constitute a simple model within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.     

Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.     

Dynamic site heterogeneity in amorphous maltose and maltitol from spectral heterogeneity in erythrosin B phosphorescence

We have used phosphorescence from erythrosin B (tetraiodofluorescein) dispersed in thin films of either maltose or maltitol to investigate the physical properties of these amorphous pure sugar matrixes. Intensity decays collected as a function of emission wavelength over the range from 640 to 720 nm were analyzed using a stretched exponential kinetic model in which the lifetime (tau) and the stretching exponent (beta) were the physically relevant parameters. The lifetimes varied systematically with emission wavelength in both matrixes. Analysis of the temperature dependence of the lifetime at each wavelength provided an estimate of the activation energy for nonradiative quenching of the triplet state; the activation energy also varied with emission wavelength. In addition, time-resolved emission spectra exhibited a blue shift with time following excitation. These data support a photophysical model in which probes are distributed among sites that vary in terms of overall molecular mobility and in which sites with lower rates of dipolar relaxation also have lower rates of collisional quenching of the erythrosin triplet state. The amorphous matrix of both maltose and maltitol in both the glass and the melt state is thus characterized by dynamic site heterogeneity in which different sites vary in terms of their overall molecular mobility.     

Thermal luminescence spectra of polyamides and their Maillard reaction with reducing sugars

Thermal luminescence (TL) spectra of polyamides were measured with a Fourier‐transform chemiluminescence spectrometer to elucidate the emission mechanism. A TL band of ε‐polylysine with a peak at 542 nm observed at 403 K was assigned to the emission due to the interaction of the –CO–NH– group with oxygen molecules by comparison with nylon‐6, polyglycine, and polyalanine. When the sample was kept at 453 K, the intensity of the TL band decreased and the wavelength of the peak shifted to 602 nm, which was assigned to the emission due to the interaction of the NH₂ group on the side chain with oxygen molecules by comparison with monomeric lysine. A weak emission with a peak at 668 nm was assigned to the advanced glycosylation end products (AGEs) yielded by the Maillard reaction with a catalytic amount of water. To understand this reaction and to examine the TL emission of AGEs, we measured TL spectra of mixtures of polylysine and reducing sugars such as glucose, maltose, lactose, and dextrin. The minimum temperature for TL emission, wavelength of the peak and the relative intensities of the TL emission were found to depend on the size of the sugars. Copyright © 2011 John Wiley & Sons, Ltd.     

An unusual red-edge excitation and time-dependent Stokes shift in the single tryptophan mutant protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers

The fluorescence emission of the single tryptophan (W233) of the mutant protein DD-carboxypeptidase from streptomyces is characterized by a red-edge excitation shift (REES), i.e., the phenomenon that the wavelength of maximum emission depends on the excitation wavelength. This phenomenon is an indication for a strongly reduced dynamic environment of the single tryptophan, which has a very low accessibility to the solvent. The REES shows, however, an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states that can be identified using the Dead-End Elimination method. The three individual lifetimes increase with increasing emission wavelength, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time-resolved anisotropy measurements that show dynamics within the rotamers but not among the rotamers. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states with different emission spectra and an anisotropic environment.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

What causes the variation of polarization degree across the emission spectrum of proteins?

A gradual decrease in fluorescence polarization across the emission spectrum on increase in wavelength has been recorded for a number of proteins and also for tryptophan, N-acetyltryptophan and glycyltryptophan. Various factors responsible for this dependence have been analyzed. It is shown that if the emission originates from both the 1La and 1Lb states, the position and form of the fluorescence spectrum polarization components as well as the slope of the dependence of the degree of polarization upon emission wavelength must always vary with the excitation wavelength. However, this condition, although necessary, is not enough to prove the participation of 1Lb in emission. The dependence of the form of the emission polarization spectrum upon excitation wavelength obtained for some proteins is explained by tyrosine residues contributing to the emission. Consequently, there are no reasons for assuming that the 1Lb oscillator participates in emission. It has been observed that for individual emitting centres, the slope of the dependence of the degree of polarization upon emission wavelength is determined by alteration of the vibrational substates, between which the transition with radiation takes place. The heterogeneity in the microenvironment properties of separate tryptophan residues in multitryptophan proteins and the existence, under certain conditions, of a correlation between the radiative lifetime of the emitting centre (determining the degree of the emission polarization) and the completeness of the microenvironment orientational relaxation (determining the emitted quantum of energy) can also affect the slope of this dependence.     

Proof of nanosecond timescale relaxation in apomyoglobin by phase fluorometry

Apomyoglobin was labeled with the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). Apparent phase shift and demodulation lifetimes of bound TNS were measured at various emission wavelengths. The lifetimes increased with increasing wavelength. Similar results were obtained for TNS in the viscous solvent glycerol at 10°C but not for TNS in vitrified or fluid solvent. The wavelength-dependent lifetimes suggest apomyoglobin is relaxing around the TNS molecule during its fluorescent lifetime. Importantly, the apparent phase lifetimes exceeded the apparent modulation lifetimes on the long wavelength side of the emission for TNS in apomyoglobin at 3°C and for TNS in glycerol at 10°C. This result proves the increasing lifetimes are a result of an excited state reaction during the lifetime of the excited state and are not a result of heterogeneity in the fluorescence emission. From the lifetimes on the short wavelength side of the emission the relaxation time of apomyoglobin was estimated to be 18 nsec.     

Erythrosin B phosphorescence monitors molecular mobility and dynamic site heterogeneity in amorphous sucrose

Molecular mobility modulates the chemical and physical stability of amorphous biomaterials. This study used steady-state and time-resolved phosphorescence of erythrosin B to monitor mobility in thin films of amorphous solid sucrose as a function of temperature. The phosphorescence intensity (lifetime), emission energy, and red-edge excitation effect were all sensitive to localized molecular mobility on the microsecond timescale in the glass and to more global modes of mobility activated at the glass transition. Blue shifts in the emission spectrum with time after excitation and systematic variations in the phosphorescence lifetime with wavelength indicated that emission originates from multiple sites ranging from short lifetime species with red-shifted emission spectrum to long lifetime species with blue-shifted emission spectrum; the activation energy for nonradiative decay of the triplet state was considerably larger for the blue-emitting species in both the glass and the melt. This study illustrates that phosphorescence from erythrosin B is sensitive both to local dipolar relaxations in the glass as well as more global relaxations in the sucrose melt and provides evidence of the value of phosphorescence as a probe of dynamic site heterogeneity as well as overall molecular mobility in amorphous biomaterials.     

Intrinsic fluorescence of the bacterial copper-containing protein amicyanin

The fluorescence properties of the single tryptophanyl residue present in amicyanin from Thiobacillus versutus are very similar to those of azurin from Pseudomonas aeruginosa and other mononuclear blue copper proteins. The emission maximum is well structured and centered at 318 nm. The quantum yield is strongly affected by the presence of copper, the removal of which is accompanied by a more than sixfold increase in fluorescence, without change in shape. The fluorescence decay of holo-amicyanin is heterogeneous with a longer component of 5.7 ns and a shorter one of 0.7 ns accounting for 90% of the total emitting molecules. Copper-free amicyanin shows instead a single exponential decay (3.3 ns) of intrinsic fluorescence. This lifetime decreases as the temperature increases as does the longer lifetime component of holoamicyanin.     

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.     

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.     

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.     

Studies on Chromophore Coupling in Isolated Phycobiliproteins: II. Picosecond Energy Transfer Kinetics and Time-Resolved Fluorescence Spectra of C-Phycocyanin from Synechococcus 6301 as a Function of the Aggregation State

The fluorescence kinetics of C-Phycocyanin in the monomeric, trimeric, and hexameric aggregation states has been measured as a function of the emission wavelength with picosecond resolution using the single-photon timing technique. All the decay curves measured at the various emission wavelengths were analyzed simultaneously by a global data analysis procedure. A sum of four exponentials was required to fit the data for the monomers and trimers. Only in the case of the hexamers, a three-exponential model function proved to be nearly sufficient to describe the experimental decays. The lifetime of those fluorescence components reflecting energy transfer decreased with increasing aggregation. This is due to the increased number of efficient acceptor molecules next to a donor in the higher aggregates. In all aggregates the shortest-lived component, ranging from 50 ps for monomer to 10 ps for hexamers, is observed as a decay term (positive amplitude) at short emission wavelength. At long emission wavelength it turns into a rise term (negative amplitude). The lifetime of a second ps-component ranges from 200 ps for monomers to 50 ps for hexamers. The long-lived (ns) fluorescence is inhomogeneous in monomers and trimers, showing two lifetimes of ~0.6 and 1.3 ns. The latter one carries the larger amplitude. The amplitudes of the kinetic components in the fluorescence decays are presented as time-resolved component spectra. A theoretical model has been derived to rationalize the observed fluorescence kinetics. Using symmetry arguments, it is shown that the fluorescence kinetics of C-Phycocyanin is expected to be characterized by three exponential kinetic components, independent of the aggregation state. An analytical expression is derived, which allows us to gain a detailed understanding of the origin of the different kinetic components and their associated time-resolved spectra. Numerical calculations of time-resolved spectra are compared with the experimental data.     

The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B

Phosphorescence from the triplet probe erythrosin B provides spectroscopic characteristics such as emission energy and lifetime that are specifically sensitive to molecular mobility of the local environment. This study used phosphorescence of erythrosin B to investigate how variation in NaCl content modulated the mobility of the amorphous sucrose matrix over the temperature range from 5 to 100 degrees C. Addition of NaCl increased the emission energy and the energy difference with excitation at the absorption maximum and the red edge, and increased the lifetime by reducing the non-radiative decay rate in the glass as well as in the undercooled liquid in a concentration dependent manner, indicating that NaCl decreased the matrix molecular mobility. Emission energy and lifetime increased with increasing NaCl content up to a maximum at NaCl/sucrose mole ratio of approximately 0.5; above 0.5 mole ratio, the effect of NaCl was less significant and appeared to be opposed by increasing plasticization by residual water. Changes in the width of the distribution of the emission energy and lifetime and variation in the lifetime with excitation and emission wavelength indicated that NaCl increased the spectral heterogeneity and thus increased the extent of dynamic site heterogeneity. These results are consistent with a physical model in which sodium and chloride ions interact with sucrose OH by ion-dipole interactions, forming clusters of less mobile molecules within the matrix.     

Electron transfer between cytochrome a and copper A in cytochrome c oxidase: a perturbed equilibrium study

Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. We have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser (0.6-ns, 1.0-mJ pulses) to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 +/- 2000 s-1 (1 sigma), at pH 7.0 and 25.5 degrees C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome a and copper A is far faster than any rate measured or inferred previously for this process.     

Multiple structures and functions of cytochrome oxidase

X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low-temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by approximately 3.8 A. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with greater than or equal to 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.     

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Observation of a stable intermediate form in the reaction of human hemoglobin with carbon monoxide

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e. 426 nm). As previously reported by others [Gray, R.D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176-5178], in the presence of 0.1 M phosphate pH 7.0 a rise-and-fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i.e. final [CO]/[heme] less than or equal to 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Y less than or equal to 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Y approximately 0.4 has a value similar to that observed starting from the fully liganded form. These results can be accounted for by a two-state allosteric model [Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the T0-R spectral changes but not for the T0-T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable.