Torsional rigidity of positively and negatively supercoiled DNA.

Time-correlated single-photon counting of intercalated ethidium bromide was used to measure the torsion constants of positively supercoiled, relaxed, and negatively supercoiled pBR322 DNA, which range in superhelix density from +0.042 to -0.123. DNA behaves as coupled, nonlinear torsional pendulums under superhelical stress, and the anharmonic term in the Hamiltonian is approximately 15 percent for root-mean-square fluctuations in twist at room temperature. At the level of secondary structure, positively supercoiled DNA is significantly more flexible than negatively supercoiled DNA. These results exclude certain models that account for differential binding affinity of proteins to positively and negatively supercoiled DNA.

A polarized photobleaching study of chromatin reorientation in intact nuclei.

Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in a compact and aggregated state which was characterized by rotational immobilization over timescales that range from 10 microseconds to 40 milliseconds. This immobilization is attributed to suppression of internal dynamics by intermolecular interactions. When the divalent cations were removed, the compact fibers no longer aggregated and were free to reorient with a characteristic decay time of about 1.2 milliseconds. It is shown that this millisecond relaxation could represent rigid rotation of topologically independent structural domains. Dilution of the monovalent salt induced a gradual change in the structural state of the chromatin that was manifest as a dramatic increase in internal flexibility. At the lowest salt concentration studied (11 mM-monovalent salt) the chromatin reorients in fewer than ten microseconds. These changes in flexibility are continuous with salt concentration, indicating that there are no well-defined endpoints to structural transitions and that the microsecond-millisecond internal dynamics of chromatin are a sensitive measure of structure. Measurements made on nuclei from cells that are either transcriptionally quiescent or active indicate that the dynamics mirrors biological activity.

The challenges of integrating molecular imaging into the optimization of cancer therapy.

We review novel, in vivo and tissue-based imaging technologies that monitor and optimize cancer therapeutics. Recent advances in cancer treatment centre around the development of targeted therapies and personalisation of treatment regimes to individual tumour characteristics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response must address spatial heterogeneity of cancer within the body. A combination of different imaging modalities should be used to relate the effect of the drug to dosing regimen or effective drug concentration at the local site of action. Molecular imaging provides a functional and dynamic read-out of cancer therapeutics, from nanometre to whole body scale. At the whole body scale, an increase in the sensitivity and specificity of the imaging probe is required to localise (micro)metastatic foci and/or residual disease that are currently below the limit of detection. The use of image-guided endoscopic biopsy can produce tumour cells or tissues for nanoscopic analysis in a relatively patient-compliant manner, thereby linking clinical imaging to a more precise assessment of molecular mechanisms. This multimodality imaging approach (in combination with genetics/genomic information) could be used to bridge the gap between our knowledge of mechanisms underlying the processes of metastasis, tumour dormancy and routine clinical practice. Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing an early read-out of response or resistance.

The renaissance of fluorescence resonance energy transfer.

Recent advances in fluorescence resonance energy transfer have led to qualitative and quantitative improvements in the technique, including increased spatial resolution, distance range, and sensitivity. These advances, due largely to new fluorescent dyes, but also to new optical methods and instrumentation, have opened up new biological applications.

Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer.

We extend the technique of fluorescence resonance energy transfer (FRET) by introducing a luminescent terbium chelate as a donor and an organic dye, tetramethylrhodamine, as an acceptor. The results are consistent with a Förster theory of energy transfer, provided the appropriate parameters are used. The use of lanthanide donors, in general, and this pair, in particular, has many advantages over more conventional FRET pairs, which rely solely on organic dyes. The distance at which 50% energy transfer occurs is large, 65 A; the donor lifetime is a single exponential and long (millisecond), making lifetime measurements facile and accurate. Uncertainty in the orientation factor, which creates uncertainty in measured distances, is minimized by the donor's multiple electronic transitions and long lifetime. The sensitized emission of the acceptor can be measured with little or no interfering background, yielding a > 25-fold improvements in the signal-to-background ratio over standard donor-acceptor pairs. These improvements are expected to make distances > 100 A measurable via FRET. We also report measurement of the sensitized emission lifetime, a measurement that is completely insensitive to total concentration and incomplete labeling.

Thiol-reactive luminescent chelates of terbium and europium.

Thiol-reactive lanthanide complexes have been synthesized that are luminescent when bound to terbium and/or europium. The complexes consist of a diethylenetriaminepentaacetate (DTPA) chelate covalently joined through one amide bond to a chromophore, carbostyril 124, and via a second amide bond to a maleimide, bromoacetamide, or pyridyldithio moiety. Site-specific attachment and characterization of the complexes attached to DNA-activating protein NtrC, to various sites on myosin, or to DNA are presented. The compounds coordinate a surprisingly large number of ligation sites of terbium when a hydrazide spacer is used between the chelate and thiol-reactive moiety, although this extra ligation can cause quenching when europium is used. Synthesis is a simple two- or three-step reaction, and purification is straightforward. The compounds should be useful as nonisotopic replacements, as long-lifetime probes in imaging, and as donors in luminescence resonance energy transfer. They are examples of a wide class of chelates that can be made conjugatable via readily available hetero- or homo-bifunctional linkers.

Amine-reactive forms of a luminescent diethylenetriaminepentaacetic acid chelate of terbium and europium: attachment to DNA and energy transfer measurements.

An isothiocyanate form of a lanthanide chelate which is highly luminescent when bound to terbium or europium has been synthesized. The chelate consists of diethylenetriaminepentaacetic acid (DTPA) covalently joined to a chromophore, 7-amino-4-methyl-2(1H)-quinolinone (cs124), and to L-p-aminophenylalanine, in which the aromatic amine was further converted to an isothiocyanate group. Ethylenediamine was also used in place of aminophenylalanine, but the isothiocyanate formed from the aliphatic amine was significantly less reactive. Site-specific attachments to triglycine and to the 5' ends of amine-modified DNA oligomers have been made. In addition, as an alternative method of coupling to macromolecules, DTPA anhydride-cs124 can be used to react specifically with a 5' amine group on base-deprotected synthetic DNA oligomers. Synthesis and purification is relatively straightforward in both cases, and luminescent properties are favorable for several applications, including as nonisotopic labels, as long-lifetime alternatives to fluorophores in imaging and diagnostics and particularly as donors in luminescence resonance energy transfer. Energy transfer measurements are consistent with previously reported measurements using different attachment mechanisms.

Quantum yields of luminescent lanthanide chelates and far-red dyes measured by resonance energy transfer.

Luminescent lanthanide chelates have unusual spectroscopic characteristics that make them valuable alternative probes to conventional organic fluorophores. However, fundamental parameters such as their quantum yield, and radiative and nonradiative decay rates have been difficult or impossible to measure. We have developed a simple and robust method based on resonance energy transfer to accurately measure these parameters. In addition, the excitation/emission process in lanthanide chelates involves several steps, and we are able to quantify each step. These include excitation of an organic antenna, transfer of energy from the antenna to lanthanide, and then lanthanide emission. Overall, the parameters show that lanthanide chelates can be efficient long-lived emitters, making them sensitive detection reagents and excellent donors in resonance energy transfer. The method is also shown to be applicable to photophysical characterization of red-emitting dyes, which are difficult to characterize by conventional means.

Luminescence resonance energy transfer measurements in myosin.

Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2 +/- 1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73 A, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5 +/- 1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.

Nanometre localization of single ReAsH molecules.

ReAsH is a red-emitting dye that binds to the unique sequence Cys-Cys-Xaa-Xaa-Cys-Cys (where Xaa is a noncysteine amino acid) in the protein. We attached a single ReAsH to a calmodulin with an inserted tetracysteine motif and immobilized individual calmodulins to a glass surface at low density. Total internal reflection fluorescence microscopy was used to image individual ReAsH molecules. We determined the centre of the distribution of photons in the image of a single molecule in order to determine the position of the dye within 5 nm precision and with an image integration time of 0.5 s. The photostability of ReAsH was also characterized and observation times ranging from several seconds to over a minute were observed. We found that 2-mercaptoethanesulphonic acid increased the number of collected photons from ReAsH molecules by a factor of two. Individual ReAsH molecules were then moved via a nanometric stage in 25 or 40 nm steps, either at a constant rate or at a Poisson-distributed rate. Individual steps were clearly seen, indicating that the observation of translational motion on this scale, which is relevant for many biomolecular motors, is possible with ReAsH.

A fluorescence photobleaching study of the microsecond reorientational motions of DNA.

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.

A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry.

The newly introduced sulfhydryl reductant tris(2-carboxyethyl)phosphine (TCEP) is a potentially attractive alternative to commonly used dithiothreitol (DTT). We compare properties of DTT and TCEP important in protein biochemistry, using the motor enzyme myosin as an example protein. The reductants equally preserve myosin's enzymatic activity, which is sensitive to sulfhydryl oxidation. When labeling with extrinsic probes, DTT inhibits maleimide attachment to myosin and must be removed before labeling. In contrast, maleimide attachment to myosin was achieved in the presence of TCEP, although with less efficiency than no reductant. Surprisingly, iodoacetamide attachment to myosin was nearly unaffected by either reductant at low (0.1 mM) concentrations. In electron paramagnetic resonance (EPR) spectroscopy utilizing nitroxide spin labels, TCEP is highly advantageous: spin labels are two to four times more stable in TCEP than DTT, thereby alleviating a long-standing problem in EPR. During protein purification, Ni(2+) concentrations contaminating proteins eluted from Ni(2+) affinity columns cause rapid oxidation of DTT without affecting TCEP. For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as EGTA in the buffer, whereas DTT is more stable if metal chelates are present. Thus TCEP has advantages over DTT, although the choice of reductant is application specific.

Temporally and spectrally resolved imaging microscopy of lanthanide chelates.

The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.

Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy.

Voltage-gated ion channels are transmembrane proteins that are essential for nerve impulses and regulate ion flow across cell membranes in response to changes in membrane potential. They are made up of four homologous domains or subunits, each of which contains six transmembrane segments. Studies of potassium channels have shown that the second (S2) and fourth (S4) segments contain several charged residues, which sense changes in voltage and form part of the voltage sensor. Although these regions clearly undergo conformational changes in response to voltage, little is known about the nature of these changes because voltage-dependent distance changes have not been measured. Here we use lanthanide-based resonance energy transfer to measure distances between Shaker potassium channel subunits at specific residues. Voltage-dependent distance changes of up to 3.2 A were measured at several sites near the S4 segment. These movements directly correlated with electrical measurements of the voltage sensor, establishing the link between physical changes and electrical charge movement. Measured distance changes suggest that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage. These results demonstrate the first in situ measurement of atomic scale movement in a trans-membrane protein.

A polarized photobleaching study of DNA reorientation in agarose gels.

Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that "reversible" photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.

Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: the effect of nucleotides and actin.

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin's enzymology. The distance between these sites is 66.8 +/- 2.3 A when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (-1.6 +/- 0.3 A) and more significantly with ADP-AlF4 (-4.6 +/- 0.2 A). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP.Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP approximately S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 approximately S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.