A spectroscopic method to determine the activity of the restriction endonuclease EcoRV that involves a single reaction.

A one-step protocol is presented to determine the activity of EcoRV as a model of restriction enzymes. The protocol involved a molecular beacon as DNA substrate, with the target sequence recognized by EcoRV in the stem. EcoRV cleaved the stem forming two fragments, one of which contained the fluorophore and quencher, initially bound by 3 bp. This shorter fragment rapidly dissociated at 37 °C, causing an increase of fluorescence intensity that was used to gauge the reaction kinetics. The reaction can be described using the Michaelis-Menten mechanism, and the kinetic parameters obtained were compared with literature values involving other protocols.

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.

Implementation of Various Modalities of the Optical-Optical Double Resonance Techniques To Simplify the Interpretation of the Spectra of Highly Excited States of Nitric Oxide.

We combined various modalities of the optical-optical double resonance (OODR) photoionization technique to simplify the interpretation of crowded molecular spectra. To demonstrate the effectiveness of our method, we applied it to the 64000 to 65200 cm(-1) spectral region of the molecule NO, where exist the following electronic states: B (2)Π (v = 21), D (2)Σ(+) (v = 5), F (2)Δ (v = 1), L (2)Π (v = 3), and K (2)Π (v = 0). This spectral region is complicated because (1) several electronic states are close in energy, (2) some of the rotational energy patterns are irregular, and (3) the relative intensity of the different bands varies markedly. We implemented four modalities of the OODR experimental technique that involved the combined use of two or three lasers. The individual rotational levels up to N' = 20 of the A(2)Σ(+) (v = 0) state were pumped as intermediate states by one-photon excitation from appropriate rotational levels in the X(2)Π (v = 0) ground state. Some of the schemes implemented provided information about line positions and relative band intensities, whereas the ion-dip detection scheme provided insight into the fate of the population in the different states. The term values that we derived are in good agreement with the literature ones. We rotationally resolved the spectra for the K (2)Π (v = 0) and B (2)Π (v = 21) states up to N = 20, and for the D (2)Σ(+) (v = 5) and L (2)Π (v = 3) states up to N = 8 and 7, respectively. Strangely, only in the rotational levels between N = 6 and N = 20 were we able to observe the F (2)Δ state, which is mostly mixed with the B' (2)Δ (v = 4) state and usually notated as F (2)Δ (v = 1) → B' (2)Δ (v = 4). We obtained the rotational constants for the B (2)Π1/2 (v = 21), L (2)Π3/2 (v = 3), and K (2)Π1/2 (v = 0) states, which had not been previously reported.

A method to estimate the elastic energy stored in braided DNA molecules using hydrodynamic equations.

We present a single-molecule method for measuring the torque exerted by braided DNA molecules undergoing spontaneous unbraiding while attached to a paramagnetic dumbbell in the absence of external manipulation. A magnetic tweezers setup is employed to braid pairs of lambda DNA molecules covalently bound to a surface. Upon removing the magnetic field, the braided DNA molecules undergo spontaneous unbraiding, efficiently transforming the stored elastic energy into enough mechanical energy to rotate the tethered dumbbells for periods as long as 30 minutes. Using hydrodynamic equations we estimate the torque exerted on the dumbbells by the DNA braids, yielding values ranging from 47 to 166 pN nm.

Structure and dynamics of single DNA molecules manipulated by magnetic tweezers and or flow.

Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules. Finally, we present some past successful examples of using these approaches to elucidate unique information about protein-nucleic acid interactions.

Rotational structure of a super-excited state of the NO molecule revealed by OODR-multiphoton laser spectroscopy.

The optical-optical double resonance time of flight (OODR-TOF) spectroscopy technique was employed to examine the 65,000-66,500 cm(-1) region of the nitric oxide spectrum. In this region, we detected the following three electronic states: E (2)Sigma(+) (nu = 2) (Rydberg state), B (2)Pi (nu = 23) (valence state), and L (2)Pi (nu = 4) (valence state). The rotational structure analysis of an unexpected band in the red part of the spectra revealed the presence of a new super-excited (2)Sigma(+) Rydberg state at approximately 13.3 eV, which was populated through a three-photon transition from the intermediate A (2)Sigma(+) (nu = 0) state. This super-excited state converges to the NO (a(3)Sigma(+)) ionic state with electronic configuration (1sigma)(2)(2sigma)(2)(3sigma)(2)(4sigma)(2)(5sigma)(2)(1pi)(3)(2pi)(1)(3ssigma)(1).

Assessment of the stability and unfolding pathways of azurin from Pseudomonas aeruginosa through the combination of denaturating osmolytes.

The denaturating osmolytes urea and guanidine hydrochloride (GuHCl) interact differently with azurin from Pseudomonas aeruginosa. At room temperature, even high concentrations of urea were unable to unfold the metalloprotein: instead a "partially unfolded" intermediate state is formed. In contrast, the protein unfolded state was formed at GuHCl, concentrations above approximately 3M and the unfolded state subsequently undergoes an irreversible reaction, which was studied employing absorbance and fluorescence spectroscopic techniques. The partially unfolded protein formed in the presence of urea completely unfolds upon adding GuHCl and we report a kinetic and thermodynamic study to characterize the process, taking into consideration that the unfolded state undergoes an irreversible reaction.

Dissociation of sulfur dioxide by ultraviolet multiphoton absorption between 224 and 232 nm.

Multiphoton excitation and dissociation of SO(2) have been investigated in the wavelength range from 224 to 232 nm. Strong evidence is found for two-photon excitation to the H Rydberg state, followed by dissociation to SO + O and ionization of the SO product by absorption of a third photon. The two-photon excitation is resonantly enhanced via the C (1)B(2) intermediate state, and the two-photon yield spectrum thus bears a strong resemblance to the spectrum of this intermediate. Imaging of the O((3)P(2)), S((1)D(2)), and SO products suggests that, following dissociation of SO(2) from the H state, SO is produced in the A and B electronic states. S((1)D(2)) is produced both from two-photon dissociation of SO(2) to give S((1)D(2)) + O(2) and by single-photon dissociation of SO(+). In the former process, the O(2) is likely formed in all of its lowest three electronic states.

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols.

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.