Time-resolved Fluorescence Polarization Anisotropy Research Articles

Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components.

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of β-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

Molecular Perspective of Antigen-mediated Mast Cell Signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that Förster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.

Modulation of GPCR Function by α‐Tocopherol

The function of vitamin E (vitE) is typically ascribed to protection from oxidation, however recent reports describe its non‐antioxidant functions. To investigate the non‐antioxidant effects of vitE in GPCR signaling, we reconstituted bovine rhodopsin into 1‐stearoyl‐2‐docosohexanoyl‐phosphatidyl choline (SDPC) bilayers. The effects of 5 and 10mol% vitE on rhodopsin function were determined spectroscopically by measuring the equilibrium amount of the signaling‐competent conformation of rhodopsin (MetaII) following photoactivation. The kinetics of MetaII formation was measured by flash photolysis. Effects of vitE on rhodopsin thermal stability were determined by differential scanning calorimetry (DSC). Phospholipid acyl chain packing was determined via DPH time‐resolved fluorescence polarization anisotropy. Addition of vitE reduced both the rate of MetaII formation and the amount of MetaII formed. DSC thermograms demonstrate that vitE increases the thermostability of rhodopsin by up to 0.8°C. Model‐dependent analysis of the scan rate‐induced shift in Tm shows that vitE slows the kinetics of rhodopsin denaturation. VitE induced more constrained acyl chain packing while reducing DPH fluorescence lifetimes. These results demonstrate that high levels of vitamin E modulate lipid packing and inhibit GPCR activation in SDPC bilayers.

Time-resolved fluorescence polarization anisotropy of multimodal samples: the asphaltene case

In this work we describe an application of the time-resolved fluorescence polarization anisotropy (TRFPA) technique to the analysis of asphaltenes, complex mixtures of high-molecular weight compounds, typically present in petroleum oils. Our asphaltene samples consist of nanometer-sized polydispersed particles, whose lighter fraction showed a relatively high fluorescence quantum yield. Most of the fluorescence intensity observed from the complex sample originated from a well defined sample fraction presenting a large fluorescence yield. Consequently, the TRFPA analysis only provided the average size of more fluorescing particles, that, in our case, were the smaller ones. Larger and less fluorescing aggregates did not significantly contribute to the TRFPA signal. Hence, to overcome intrinsic limitations of the TRFPA technique in characterizing multimodal samples, we preliminarily fractionated our complex samples by means of size exclusion chromatography (SEC), thus obtaining nearly monomodal fractions of the original samples. This procedure allowed to estimate also the size of less fluorescing and larger particles. A comparison of particle size estimate by means of TRFPA and SEC methods was also used to acquire information about occurrence of aggregation phenomena, and about the kind and strength of the chemical bonds linking chromophores to each other or to their parent particle.

Evidence of fluorescent carbon nanoparticles produced in premixed flames by time-resolved fluorescence polarization anisotropy

In this work we report on the analysis of combustion-generated nano-organic carbon (NOC) particles by means of time-resolved fluorescence polarization anisotropy (TRFPA). NOC samples were collected in water from nonsooting regions of ethylene/air laminar flames. The size of collected particles was determined with subnanometer accuracy by exploiting femtosecond laser pulses as an excitation source. Moreover, the TRFPA measurements were realized by selecting narrow-wavelength bands within the fluorescence spectrum. With this procedure, the ensemble-averaged size of particles emitting in the selected fluorescence band was determined, to provide additional information on composition and spectroscopic properties of the investigated particles. In particular, the NOC samples consisted of two groups of particles, preferentially emitting in two distinct wavelength bands: smaller particles, with diameter d = 1 – 1.5 nm , mostly fluorescing in the UV ( λ ∼ 300 – 470 nm ), and larger particles, d > 2 nm , fluorescing in the visible ( λ ∼ 490 – 580 nm ). The results of this work strongly support the attribution of UV/visible fluorescence generally detected in flames to nanoparticles. They also evidence that nanoparticles undergo a chemical transformation during the growth process, which produces a red shift in the fluorescence spectrum.

Time-resolved fluorescence polarization anisotropy and optical imaging of Cybesin in cancerous and normal prostate tissues

Time-resolved polarization-dependent fluorescence of Cybesin in solution and in cancerous and normal prostate tissues were measured. The polarization preservation property of Cybesin in tissue was observed. The fluorescence intensity emitted from a Cybesin-stained cancerous tissue area was found to be much stronger than that from a Cybesin-stained normal tissue area indicating that cancerous prostate tissue takes-up more Cybesin than normal tissue. The polarization anisotropy of Cybesin contained in cancerous prostate tissue was found to be larger than that of Cybesin in normal prostate tissue indicating that a larger degree of polarization was preserved in the Cybesin-stained cancerous tissue due to structures. A static anisotropy component from the emission of cell-bonded Cybesin molecules in tissue and a time-dependent anisotropy component from the emission of un-bonded Cybesin molecules were determined and discussed. The static anisotropy value of Cybesin in stained cancerous tissue was found to be much larger than that in stained normal tissue. The fluorescence polarization difference imaging technique based on the polarization preservation of Cybesin was used to enhance the image contrast between cancerous and normal prostate tissue areas.

Effects of ethylene glycol on the torsion elastic constant and hydrodynamic radius of p30δ DNA

Upon increasing the concentration of ethylene glycol (EG) at 37 degrees C, the twist energy parameter, E(T), which governs the supercoiling free energy, was recently found to undergo a decreasing (or reverse) sigmoidal transition with a midpoint near 20 w/v % EG. In this study, the effects of adding 20 w/v % EG on the torsion elastic constant (alpha) of linear p30delta DNA and on the hydrodynamic radius (R(H)) of a synthetic 24 bp duplex DNA were examined at both 40 and 20 degrees C. The time-resolved fluorescence intensity and fluorescence polarization anisotropy (FPA) of intercalated ethidium were measured in order to assess the effects of 20 w/v % EG on: (1) alpha; (2) R(H); (3) the lifetimes of intercalated and non-intercalated dye; (4) the amplitude of dye wobble in its binding site; and (5) the binding constant for intercalation. The effects of 20 w/v % EG on the circular dichroism (CD) spectrum of the DNA and on the emission spectrum of the free dye were also measured. At 40 degrees C, addition of 20 w/v % EG caused a substantial (1.27- to 1.35-fold) increase in alpha, a significant change in the CD spectrum, and a very small, marginally significant increase in R(H), but little or no change in the amplitude of dye wobble in its binding site or the lifetime of intercalated dye. Together with previously reported measurements of E(T), these results imply that the bending elastic constant of DNA is significantly decreased by 20 w/v % EG at 40 degrees C. At 20 degrees C, addition of 20 w/v % EG caused a marginally significant decrease in alpha and very little change in any other measured properties. Also at 20 degrees C, addition of 30 w/v % betaine caused a marginally significant increase in alpha and significant but modest change in the CD spectrum, but very little change in any other properties.

Characterization of ultrafast fluorescence from nanometric carbon particles

We present two distinct implementations of the time resolved fluorescence polarization anisotropy (TRFPA) technique for the analysis of carbon nanoparticles collected from laminar laboratory flames and from the exhaust of a gasoline vehicle engine. By exploiting the high time-resolution and the spectral resolution of our TRFPA setups, we could identify two groups of particles, with diameter of 1.4 and 2.2 nm, respectively, within the laminar flame sample. On the other hand, the high time-resolution TRFPA analysis of the gasoline sample led to the identification of two distinct decay channels. The slower one was consistent with a single kind of small particle with diameter of 1.3 nm. Moreover, an analysis of the fast decay versus the temperature allowed us to rule out that it was due to the presence of lighter particles within the sample. Most likely, it is related to the relaxation of internal degrees of freedom.

Characterization of nanometric carbon materials by time-resolved fluorescence polarization anisotropy

In this work, time-resolved fluorescence polarization anisotropy (TRFPA) technique has been applied to the determination of the average size of asphaltenes and combustion-generated carbon nanoparticles. The characteristic depolarization time of fluorescence light following photon absorption is related to the typical particle size through the Stoke–Einstein rotational diffusion equation. The TRFPA technique employed in our experiment achieves sub-nanosecond time resolution, roughly corresponding to sub-nanometer accuracy in determining the particle size. The technique has been applied to both asphaltene and carbon nanoparticles, the former being a component of petroleum, whereas the latter result from combustion processes. Therefore, a complete and reliable characterization of such particles is of great importance in oil industry and atmospheric physics, respectively. Although the TRFPA technique has been developed and widely used on molecular and biological samples, it proves to be a very powerful tool for measuring the size of asphaltene and soot particles as small as few nanometers with a resolution of the order of 0.1 nm.

Aggregation and interactions of C 60 and C 70 fullerenes in neat N-methylpyrrolidinone and in N-methylpyrrolidinone/toluene mixtures

A strong solvatochromic effect leading to the loss of the fine structure of UV–visible spectra and to the change of the specific absorption was observed for C 60 and C 70 fullerenes in N-methylpyrrolidinone (NMP). The C 60 and C 70 fullerene interactions in NMP solutions were studied by using time resolved fluorescence polarization anisotropy and dynamic light scattering techniques aiming to the determination of fullerene sizes. The process was studied as time dependent in toluene/NMP solutions and both the formation of fullerenes aggregates and specific interactions between fullerenes and NMP were observed.

Time resolved fluorescence polarization anisotropy of carbonaceous particles produced in combustion systems

The size of nanometric carbonaceous particles produced in various combustion systems is determined by means of time resolved fluorescence polarization anisotropy (TRFPA). We also compare the performances of two different experimental implementations of thetechnique, which are complementary in terms of cost, simplicity and resolution. Both methods are first employed on standard molecules to demonstrate the reliability of the results. A study of the sizes of nanometric particles collected at the exhaust of diesel and gasoline vehicle engine, as well as from controlled laminar flames is presented. The high sensitivity (0.04 nm) achieved with the use of a streak camera as detector makes the TRFPA technique particularly suitable for characterizing nanometric particles.

Effects of small neutral osmolytes on the supercoiling free energy and intrinsic twist of p30delta DNA.

Both theory and experiments are employed to investigate the effects of small neutral osmolytes on the average intrinsic twist (l0), the torsion and bending elastic constants, and the twist energy parameter (ET) that governs the supercoiling free energy. The experimental data for ethylene glycol and acetamide at 37 degrees C suggest, and are interpreted in terms of, a model wherein the DNA exhibits an equilibrium between two distinct conformational states that possess different numbers of bound water molecules and exhibit different intrinsic twists and torsion and bending elastic constants. Expressions are derived to relate the effective ET and l0 to the equilibrium constant, water activity (aw), and number (n) of bound water molecules released per cooperative domain undergoing the two-state transition. The variations of l0 and ET with -ln(aw) are similar for acetamide and ethylene glycol at 37 degrees C. Fitting the theory to those data yields the range n = 103-125 for ethylene glycol and n = 71-113 for acetamide, depending on the assumed value of ET for the dehydrated state. The cooperative domain size of the two-state transition is estimated to exceed about 25-30 base pairs (bp). Between 0 and 19.4 w/v % ethylene glycol, the torsion elastic constant, measured by time-resolved fluorescence polarization anisotropy (FPA), increases by 1.37-fold, whereas the measured ET decreases by 1.15-fold over that same range. The implied decrease in bending rigidity over that range is by a factor of about 0.7. The variations of l0 and ET with increasing -ln(aw) due to added ethylene glycol at 37 degrees C are far smaller than the corresponding variations observed previously at 14 and 15 degrees C. However, at 21 degrees C, upon adding either ethylene glycol or acetamide, l0 and ET initially decline steeply with increasing -ln(aw), with slopes possibly comparable to those seen at 14 and 15 degrees C, but then flatten out and follow curves similar to those at 37 degrees C. Possible origins of such mixed behavior are discussed. The effects of betaine at both 37 and 21 degrees C differ qualitatively and quantitatively in various respects from those of ethylene glycol and acetamide. Upon adding sucrose, l0 initially jumps to higher plateaus at both 37 and 21 degrees C, but its effects on ET cannot be reliably assessed, due to the limited range of -ln(aw).

Molecular dynamics of guest molecules in micelles: models based on fluorescence polarization dynamics

Time-resolved fluorescence polarization (anisotropy) of a probe (guest molecule) in a micelle is used for testing different models of molecular dynamics. The experimental studies so far support the model that includes wobbling motion and translational diffusion for the guest molecule in the micelle.

Dynamic Bending Rigidity of a 200-bp DNA in 4 mM Ionic Strength: A Transient Polarization Grating Study

DNA may exhibit three different kinds of bends: 1) permanent bends; 2) slowly relaxing bends due to fluctuations in a prevailing equilibrium between differently curved secondary conformations; and 3) rapidly relaxing dynamic bends within a single potential-of-mean-force basin. The dynamic bending rigidity ( κ d), or equivalently the dynamic persistence length, P d = κ d /k B T, governs the rapidly relaxing bends, which are responsible for the flexural dynamics of DNA on a short time scale, t ≤ 10 −5 s. However, all three kinds of bends contribute to the total equilibrium persistence length, P tot, according to 1/P tot ≅ 1/P pb + 1/P sr + 1/P d, where P pb is the contribution of the permanent bends and P sr is the contribution of the slowly relaxing bends. Both P d and P tot are determined for the same 200-bp DNA in 4 mM ionic strength by measuring its optical anisotropy, r( t) , from 0 to 10 μs. Time-resolved fluorescence polarization anisotropy (FPA) measurements yield r( t) for DNA/ethidium complexes (1 dye/200 bp) from 0 to 120 ns. A new transient polarization grating (TPG) experiment provides r( t) for DNA/methylene blue complexes (1 dye/100 bp) over a much longer time span, from 20 ns to 10 μs. Accurate data in the very tail of the decay enable a model-independent determination of the relaxation time ( τ R) of the end-over-end tumbling motion, from which P tot = 500 Å is estimated. The FPA data are used to obtain the best-fit pairs of P d and torsion elastic constant ( α) values that fit those data equally well, and which are used to eliminate α as an independent variable. When the relevant theory is fitted to the entire TPG signal ( S( t)) , the end-over-end rotational diffusion coefficient is fixed at its measured value and α is eliminated in favor of P d. Neither a true minimum in chi-squared nor a satisfactory fit could be obtained for P d anywhere in the range 500–5000 Å, unless an adjustable amplitude of azimuthal wobble of the methylene blue was admitted. In that case, a well-defined global minimum and a reasonably good fit emerged at P d = 2000 Å and 〈 δζ 2〉 1/2 = 25°. The discrimination against P d values <1600 Å is very great. By combining the values, P tot = 500 Å and P d = 2000 Å with a literature estimate, P pb = 1370 Å, a value P sr = 1300 Å is estimated for the contribution of slowly relaxing bends. This value is analyzed in terms of a simple model in which the DNA is divided up into domains containing m bp, each of which experiences an all-or-none equilibrium between a straight and a uniformly curved conformation. With an appropriate estimate of the average bend angle per basepair of the curved conformation, a lower bound estimate, m = 55 bp, is obtained for the domain size of the coherently bent state. Previous measurements suggest that this coherent bend is not directional, or phase-locked, to the azimuthal orientation of the filament.

Effect of polyethylene glycol on the supercoiling free energy of DNA.

The supercoiling free energy of pUC19 DNA [2686 base pairs (bp)] was measured in various concentrations of PEG 8000 (polyethylene glycol; molecular weight 8000) by the topoisomer distribution method. The effective twist energy parameter (E(T)) that governs the supercoiling free energy declined linearly by 1.9-fold with increasing w/v % PEG from 0 to 7.5%, which lies below the threshold for intermolecular condensation. In principle, PEG could affect E(T) either via an osmotic exclusion mechanism or by altering the torsion elastic constant, bending rigidity, or self-repulsions of the DNA. Possible alterations of the DNA secondary structure and torsion elastic constant were assessed by CD spectroscopy and time-resolved fluorescence polarization anisotropy of intercalated ethidium. Up to 7.5% PEG, the secondary structure of the DNA remained largely unaltered, as evidenced by (1) the absence of any significant change in the CD spectrum, (2) an extremely small relative decrease (-0.0013) in intrinsic twist, and (3) a negligibly small change in the torsion elastic constant. The observed reduction in E(T) cannot be ascribed primarily to a decrease in torsion elastic constant, and most likely does not stem from a decrease in bending rigidity either. The decrease in medium dielectric constant due to PEG should increase the self-repulsions, and thereby increase E(T), which is opposite to the observed trend. Instead, the observed decline in E(T) is attributed to an osmotic exclusion mechanism. The change in molar volume excluded to the PEG (Delta V(ex)), when the linking difference converts from Delta l = 0 to Delta l = +/-1, was determined from the observed E(T) value and PEG osmotic pressure at each concentration. The experimental Delta V(ex) values agree well with theoretical estimates reckoned for a simple osmotic exclusion model, in which PEG is excluded by hard-core interactions from a concentric cylindrical volume around every duplex segment. The difference in volume excluded to PEG between the Delta l = 0 and the Delta l = +/-1 topoisomers is attributed entirely to the approximately 0.7 additional writhe "crossing" of two duplex strands at roughly 90 degrees, which is known to occur in the latter species. When the separation between the duplex centers at the "crossing" was adjusted so that the theoretical estimate of Delta V(ex) matched the experimental value at each PEG concentration, a value near 5.7 nm was obtained in each case. The invariance and plausible magnitude of this mean separation at the crossing provide strong support for this simple osmotic exclusion model. An alternative model, in which the PEG is excluded from the entire coil envelope of the DNA out to its radius of gyration, perhaps because it decreases the local dielectric constant, was also considered. The estimated difference in excluded volume in that case exceeds the experimental value by a factor of nearly 10(4), and could be ruled out on that basis.

Phase Separation in Binary and Ternary Polymer Composites Studied with Electronic Excitation Transport

Time-resolved fluorescence polarization anisotropy decay measurements are used to observe the process of phase separation of molecularly mixed polymer blends. The binary blends are composed of a low concentration of poly(methyl methacrylate-co-2-vinylnaphthalene) (PMMA2VN) in poly(vinyl acetate) (PVAc). The small number of 2VN groups on the PMMA2VN chains act as fluorescent chromophores. Electronic excitation transport (EET) among these chromophores contributes to the decay of their fluorescence polarization anisotropy, which is measured with time-correlated single photon counting. Since EET is highly sensitive to the distances between chromophores, it reflects their spatial distribution and can reveal both the isolated-chain structure and the aggregation of polymer chains in the blend on the nanometer distance scale. Blend samples are prepared by annealing a homogeneous blend at an elevated temperature for a given time and then rapidly quenching the phase-separating material below its glass-transition temperature. The newly formed nanoscopic aggregates frozen in the sample are then investigated with EET. The ternary blends are composed of a very small amount of PMMA2VN and a larger quantity of PMMA, both in bulk PVAc. In the phase separation of ternary blends, the PMMA2VN component initially partitions with its chromophore-free counterpart, expanding to its i-condition size before continuing to aggregate and eventually separating from the PMMA.

Effect of Lipid Molecules on Twisting Motions of DNA Helix Studied by Fluorescence Polarization Anisotropy

Time-resolved fluorescence polarization anisotropy (FPA) of two dyes (acridine orange and ethidium bromide) intercalated in a raw DNA and a novel DNA-lipid complex has been measured on nanosecond timescale to evaluate the effect of lipid molecules on the twisting motions of DNA helix. Steady-state fluorescence spectra show that intercalated dyes are not influenced much by solvents compared with free dyes, suggesting the similar microscopic environments of intercalated chromophores. The FPA decay times of both dyes are about twice larger in the DNA-lipid complex than in the raw DNA, which indicates that the lipid molecules around the helix make the twisting motion slower. The FPA decay profiles fit very well with double-exponential functions having time constants differing much with each other, suggesting that there exists slow and quick elements in the twisting motions.

Effect of temperature on DNA secondary structure in the absence and presence of 0.5 M tetramethylammonium chloride.

Changes in the average secondary structures of three different linear DNAs over the premelting region from 5 to 60 degrees C were investigated by measuring their CD spectra and also their torsion elastic constants (<alpha>) by time-resolved fluorescence polarization anisotropy. For one of these DNAs, the Haell fragment of pBR322, the apparent diffusion coefficients [Dapp(k)] at small and large scattering vectors (k) were also measured by dynamic light scattering. With increasing temperature, all three DNAs exhibited typical premelting changes in their CD spectra, and these were accompanied by 1.4- to 1.7-fold decreases in <alpha>. Also for the 1876 base pair fragment, Dapp(k) at large scattering vectors, which is sensitive to the dynamic bending rigidity, decreased by 17%, even though there was no change at small scattering vectors, where Dapp(k) = D0 is the translational diffusion coefficient of the center-of-mass. These observations demonstrate conclusively that the premelting CD changes of these DNAs are associated with a significant change in average secondary structure and mechanical properties, though not in persistence length. In the presence of 0.5 M tetramethylammonium chloride (TMA-Cl) the premelting change in CD is largely suppressed, and the corresponding changes in <alpha> and Dapp(k) at large scattering vectors are substantially diminished. These observations suggest that TMA-Cl, which binds preferentially to A.T-rich regions and stabilizes those regions (relative to G.C-rich regions) against melting, effectively stabilizes the prevailing low-temperature secondary structure sufficiently that the DNA is effectively trapped in that state over the temperature range observed.

On the origin of the temperature dependence of the supercoiling free energy

Monte Carlo simulations using temperature-invariant torsional and bending rigidities fail to predict the rather steep decline of the experimental supercoiling free energy with increasing temperature, and consequently fail to predict the correct sign and magnitude of the supercoiling entropy. To illustrate this problem, values of the twist energy parameter (E(T)), which governs the supercoiling free energy, were simulated using temperature-invariant torsion and bending potentials and compared to experimental data on pBR322 over a range of temperatures. The slope, -dE(T)/dT, of the simulated values is also compared to the slope derived from previous calorimetric data. The possibility that the discrepancies arise from some hitherto undetected temperature dependence of the torsional rigidity was investigated. The torsion elastic constant of an 1876-bp restriction fragment of pBR322 was measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium over the range 278-323 K, and found to decline substantially over that interval. Simulations of a 4349-bp model DNA were performed using these measured temperature-dependent torsional rigidities. The slope, -dE(T)/dT, of the simulated data agrees satisfactorily with the slope derived from previous calorimetric measurements, but still lies substantially below that of Duguet's data. Models that involve an equilibrium between different secondary structure states with different intrinsic twists and torsion constants provide the most likely explanation for the variation of the torsion constant with T and other pertinent observations.

Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.