Marcia Levitus

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only ∼250 ms before spontaneously unwrapping; unwrapped DNA rewraps within ∼10–50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.

Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

Dynamics of nucleosome invasion by DNA binding proteins.

Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

Photophysics of Backbone Fluorescent DNA Modifications: Reducing Uncertainties in FRET

We have investigated the photophysical properties of backbone fluorescent DNA modifications with the goal of reducing many of the sources of uncertainty commonly encountered in Forster resonance energy transfer (FRET) measurements. We show that backbone modifications constrain rotational motions, providing a way by which the orientation of the dye can be controlled in a predictable manner, and reduce the uncertainties in donor-acceptor distance associated with the flexible linkers commonly used in conjugate chemistry. Rotational rigidity also prevents undesirable dye-DNA interactions, which have been shown to affect the photophysical properties of the dye. Unusually large FRET efficiencies for donor-acceptor pairs separated by 102 A (three helical turns) were measured and attributed to the favorable relative orientation of the dipoles. The same FRET efficiency was measured for a sample in which the donor-acceptor separation was 12 A shorter, demonstrating the important role of relative orientation in FRET experiments.

Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3

Protein-induced fluorescence enhancement (PIFE) is a term used to describe the increase in fluorescence intensity observed when a protein binds to a nucleic acid in the proximity of a fluorescent probe. PIFE using the single-molecule dye Cy3 is gaining popularity as an approach to investigate the dynamics of proteins that interact with nucleic acids. In this work, we used complexes of DNA and Klenow fragment and a combination of time-resolved fluorescence and transient spectroscopy techniques to elucidate the photophysical mechanism that leads to protein-enhanced fluorescence emission of Cy3 when in close proximity to a protein (PIFE). By monitoring the formation of the cis isomer directly, we proved that the enhancement of Cy3 fluorescence correlates with a decrease in the efficiency of photoisomerization, and occurs in conditions where the dye is sterically constrained by the protein.

DIRECT MEASUREMENT OF THE DIPOLE MOMENT OF A METASTABLE MEROCYANINE BY ELECTROMECHANICAL INTERFEROMETRY

Abstract The permanent dipole moments of an indolinespirobenzopyran and its merocyanine from in a poly(isobutyl methacrylate) host were determined by electromechanical interferometry. This technique allows the determination of the piezoelectric and electrostrictive properties of thin films for which the polarization of the sample can be determined. The sample was exposed to an external electric field and the polarization was measured as a function of time before and after UV irradiation. Results show that the orientation of spiropyran and merocyanine molecules in the direction of the field is fast compared with the time scale of the photoisomer decay kinetics, allowing the determination of dipole moments from polarization values in the limit of thermal equilibrium. The experiments reveala pronounced increase in the dipole moment upon ring opening with μ0=6.4 D for the spirobenzopyran and μ0=14.2 D for the merocyanine form.

FRET fluctuation spectroscopy of diffusing biopolymers: contributions of conformational dynamics and translational diffusion.

The use of fluorescence correlation spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in fluorescence resonance energy transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments. Results show that diffusion does not contribute to the measured fluctuations in FRET efficiency in conditions where the relaxation time of the kinetic process is much shorter than the mean transit time of the molecules in the optical observation volume. However, in contrast to what has been suggested in previous work, the contributions of diffusion are otherwise significant. Neglecting the contributions of diffusion can potentially lead to an erroneous interpretation of the kinetic mechanisms. As an example, we demonstrate that the analysis of FRET fluctuations in terms of a purely kinetic model would generally lead to the conclusion that the system presents complex kinetic behavior even for an idealized two-state system.

Photophysical and Dynamical Properties of Doubly Linked Cy3 - DNA Constructs

Photophysical measurements are reported for Cy3-DNA constructs in which both Cy3 nitrogen atoms are attached to the DNA backbone by short linkers. While this linking was thought to rigidify the orientation of the dye and hinder cis-isomerization, the relatively low fluorescence quantum yield and the presence of a short component in the time-resolved fluorescence decay of the dye indicated that cis-isomerization remained possible. Fluorescence correlation spectroscopy and transient absorption experiments showed that photoisomerization occurred with high efficiency. Molecular dynamics simulations of the trans dye system indicated the presence of stacked and unstacked states, and free energy simulations showed that the barriers for stacking/unstacking were low. In addition, simulations showed that the ground cis state was feasible without DNA distortions. Based on these observations, a model is put forward in which the doubly linked dye can photoisomerize in the unstacked state.

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Probing the Interaction Between Fluorophores and DNA Nucleotides by Fluorescence Correlation Spectroscopy and Fluorescence Quenching

We have investigated the association interactions between the fluorescent dyes TAMRA, Cy3B and Alexa‐546 and the DNA deoxynucleoside monophosphates by means of fluorescence quenching and fluorescence correlation spectroscopy (FCS). The interactions of Cy3B and TAMRA with the nucleotides produce a decrease in the apparent diffusion coefficient of the dyes, which result in a shift toward longer times in the FCS autocorrelation decays. Our results with Cy3B demonstrate the existence of Cy3B‐nucleotide interactions that do not affect the fluorescence intensity or lifetime of the dye significantly. The same is true for TAMRA in the presence of dAMP, dCMP and dTMP. In contrast, the diffusion coefficient of Alexa 546 remains practically unchanged even at high concentrations of nucleotide. These results demonstrate that interactions between this dye and the four dNMPs are not significant. The presence of the negatively charged sulfonates and the bulky chlorine atoms in the phenyl group of Alexa 546 possibly prevent strong interactions that are otherwise possible for TAMRA. The characterization of dye–DNA interactions is important in biophysical research because they play an important role in the interpretation of energy transfer experiments, and because they can potentially affect the structure and dynamics of the DNA.