Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Photochemical reactions of LAC repressor. Effects on inducer binding.

Insulin was tritiated by exposure to tritium gas activated by microwave radiation. ³H-insulin competed with ¹²⁵I-insulin for binding to cultured human lymphocytes and to anti-insulin antibody to the same extent as did native insulin. The affinity constant for the binding of ³H-insulin to specific receptors on cultured human lymphocytes was 0.48 × 10⁹ M^?1 (SD-0.06). The affinity constant for the binding of ¹²⁵I-insulin was 0.57 × 10⁹ M^?1 (SD=0.23). As was the case with ¹²⁵I-insulin, the Scatchard plot of the binding of ³H-insulin to human lymphocytes was curvilinear, suggesting the presence of a heterogeneous population of receptors, or of a homogeneous population of receptors that exhibit negative cooperativity. The similarity observed between ³H-insulin and ¹²⁵I-insulin helps refute the argument that distortion of the insulin molecule caused by introduction of an iodine atom may interfere with its binding to insulin receptors.     

Binding of E.coli lac repressor to non-operator DNA*

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] 相似文献   

Resolution and characterization of tryptophyl fluorescence of hen egg-white lysozyme by quenching- and time-resolved spectroscopy

The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region.     

Dynamics of the peptide hormone motilin studied by time resolved fluorescence spectroscopy

Time resolved fluorescence was used to study the dynamics on the nanosecond and subnanosecond time scale of the peptide hormone motilin. The peptide is composed of 22 amino acid residues and has one tyrosine residue in position 7, which was used as an intrinsic fluorescence probe. The measurements show that two rotational correlation times, decreasing with increasing temperature, are needed to account for the fluorescence polarization anisotropy decay data. Viscosity measurements combined with the fluorescence measurements show that the rotational correlation times vary approximately as viscosity with temperature. The shorter rotational correlation time (0.08 ns in an aqueous solution with 30% hexafluoropropanol, HFP at 20°C) should be related to internal movement of the tyrosine side chain in the peptide while the longer rotational correlation time (2.2 ns in 30% HFP at 20°C) describes the motion of the whole peptide. In addition, the interaction of motilin or the derivative motilin (Y7F) –23W (with tyrosine substituted by phenylalanine and with a tryptophan fluorophore added to the C-terminal) with negatively charged phospholipid vesicles (DOPG) was studied. The results show the development of a long anisotropy decay time which reflects partial immobilization of the peptide by interaction with the vesicles.Correspondence to: A. Gräslund     

E. coli trp repressor forms a domain-swapped array in aqueous alcohol

The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 A X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.     

Time resolved spectroscgpy of tryptopkyl fluorescence of yeast 3-phosphoglycerate kinase

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime τ₁= 0.6 ns and a maximum fluorescence wavelength λ_2max = 332 nm. The second tryptophan residue exhibits two lifetimes τ₂₁= 3.1 ns and τ₂₂= 7.0 ns (λ_2max= 338 nm). In agreement with the attribution of τ₂₁and τ₃₂ to the same tryptophan residue, the ratio β = C₂₁/C₂₂ of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally tc the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay times and a neighbouring protein chemical group. The intrinsic pK of this group and the equilibrium constant of the irternal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.     

Dynamic force spectroscopy of E. coli P pili

Surface organelles (so-called pili) expressed on the bacterial membrane mediate the adhesion of Escherichia coli causing urinary tract infection. These pili possess some extraordinary elongation properties that are assumed to allow a close bacterium-to-host contact even in the presence of shear forces caused by urine flow. The elongation properties of P pili have therefore been assessed for low elongation speeds (steady-state conditions). This work reports on the behavior of P pili probed by dynamic force spectroscopy. A kinetic model for the unfolding of a helixlike chain structure is derived and verified. It is shown that the unfolding of the quaternary structure of the PapA rod takes place at a constant force that is almost independent of elongation speed for slow elongations (up to approximately 0.4 mum/s), whereas it shows a dynamic response with a logarithmic dependence for fast elongations. The results provide information about the energy landscape and reaction rates. The bond length and thermal bond opening and closure rates for the layer-to-layer bond have been assessed to approximately 0.76 nm, approximately 0.8 Hz, and approximately 8 GHz, respectively. The results also support a previously constructed sticky-chain model for elongation of the PapA rod that until now had been experimentally verified only under steady-state conditions.     

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.     

Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.

In the present work, we have characterized the protein--protein interactions in the trp repressor (TR) from Escherichia coli using fluorescence spectroscopy. The steady-state and time-resolved fluorescence anisotropy of repressor labeled with 5-(dimethylamino)naphthalene-1-sulfonamide (DNS) was used to monitor subunit equilibria in the absence and presence of corepressor. In the absence of tryptophan, the repressor is in equilibrium between tetramers and dimers in the concentration range studied (approximately 0.04-40 microM in dimer). Binding of corepressor resulted in a marked destabilization of the tetramer. The beginning of a dimer-monomer dissociation transition was observed by monitoring the decrease in the intrinsic tryptophan emission energy upon dilution below 0.1 microM in dimer, indicating an upper limit for the dimer-dissociation constant near 1 nM. DNA titrations with a 26 base pair sequence containing the trp EDCBA operator performed in the absence and presence of the corepressor are consistent with a 1:1 dimer/operator stoichiometry in the presence of tryptophan, while the aporepressor binds with TR dimer/DNA stoichiometries greater than one and which depend upon both the concentration of protein and that of the operator. Using the multiple observable parameters available in fluorescence, we have thus carried out a thorough investigation of the coupled equilibria in this bacterial repressor. Our results are consistent with a physiologically relevant thermodynamic role for tetramerization in the regulatory function of the trp repressor. The present results which have brought to light novel protein--protein interactions in the trp repressor system indicate that fluorescence spectroscopic methods could prove quite useful in the study of the role of protein--protein interactions in eukaryotic systems as well.     

Alanine screening mutagenesis establishes the critical inactivating damage of irradiated E. coli lactose repressor

The function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor.     

72 residues of gal repressor fused to beta-galactosidase repress the gal operon of E. coli

An active gene has been constructed which produces a chimera consisting of the N-terminal domain of the gal repressor and all but the first five residues of beta-galactosidase. Seventy two residues of gal repressor fused to beta-galactosidase as tetrameric core are sufficient to repress the gal operon in vivo and to bind to the gal operator in vitro.     

Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.     

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.     

Analysis of inducers of the E.coli lac repressor system in mammalian cells and whole animals.

Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system. These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers. In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours. Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration. Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG. In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes. HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor. These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.     

The arginine repressor of Escherichia coli.

This review tells the story of the arginine repressor of Escherichia coli from the time of its discovery in the 1950s until the present. It describes how the research progressed through physiological, genetic, and biochemical phases and how the nature of the repressor and its interaction with its target sites were unraveled. The studies of the repression of arginine biosynthesis revealed unique features at every level of the investigations. In the early phase of the work they showed that the genes controlled by the arginine repressor were scattered over the linkage map and were not united, as in other cases, in a single operon. This led to the concept of the regulon as a physiological unit of regulation. It was also shown that different alleles of the arginine repressor could result in either inhibition of enzyme formation, as in E. coli K-12, or in stimulation of enzyme formation, as in E. coli B. Later it was shown that the arginine repressor is a hexamer, whereas other repressors of biosynthetic pathways are dimers. As a consequence the arginine repressor binds to two palindromic sites rather than to one. It was found that the arginine repressor not only acts in the repression of enzyme synthesis but also is required for the resolution of plasmid multimers to monomers, a completely unrelated function. Finally, the arginine repressor does not possess characteristic structural features seen in other prokaryotic repressors, such as a helix-turn-helix motif or an antiparallel beta-sheet motif. The unique features have sustained continuous interest in the arginine repressor and have made it a challenging subject of investigation.