Thermal denaturation profiles

Fig. 5. NMR thermal denaturation profiles of the Sso7d wild type and its PheSlAla, PheSlTyr, Tyr23Ala and Trp33Ala mutants determined by monitoring changes in H chemical shifts of the methyl resonances of Met57. Reprinted with permission from Biochemistry, Vol. 38, R. Consonni, L. Santomo, P. Fusi, P. Tortora and L. Zetta, 1999, p. 12,709. Copyright (1999) American Chemical Society.

Hillyer, G.V. (1974) Buoyant density and thermal denaturation profiles of schistosome DNA. Journal of Parasitology 60, 725-727. 

Thermal denaturation profiles were done using one of the molecular beacon structures in solution, without the alkyl chain linker attached to the 3 -thymidine residue. The fully complementary and single base pair mismatched sequence targets were investigated. The differences of Tm from the melt profiles showed that single base pair mismatches could not be discriminated from the complementary target material. Melt curves were also... 

Figure 2.21 spFRET histograms, with the zero peaks removed, of a 19 nucleotide RNA hairpin (described in the text and measured as described in Figure 2.20) at various temperatures along its thermal denaturation profile (black circles, measured by ensemble FRET). As the population changes from all folded (closed hairpins) to all unfolded (open hairpins) the dominant peak in the histograms switches from high FRET efficiency to low FRET efficiency. 

These latter studies were done in low salt at very low concentrations of poly I poly C (ca 5.0 x 10 M) and poly-L-lysine (ca 2.25 x 10 M). The complex was formed at room temperature. The poly-L-lysine used in these complexes was of a high molecular weight (60-90,000). These investigators reported the binding reaction of poly-L-lysine to polynucleotide to be quantitative, irreversible and with a definite stoichiometric ratio. The poly-L-lysine to poly I poly C ratio at these concentrations was 0.5 NH PO. The thermal de-naturation profile of this complex was found to be a one-step transition with a T at about 89°C in a solvent which contained 0.05 M-NaCl + 0.001 M-9a Citrate ( " 0.3X SSC). Complexes formed between poly I-poly C and poly-L-lysine with less poly-L-lysine than the 1P 0 5 NH ratio gave two-step thermal denaturation profiles with T s at 5rC and 89 C. The lower temperature (T ) indicated that tSere was still free poly I poly C. 

Thermal Denaturation Profiles. Thermal denaturation profile studies were conducted to determine the optimum annealing temperature for the real-time PCR. The changes in fluorescence of a 50 pi solution containing 0.3pM of the beacon probe with or without 0.9uM of a perfectly complimentary single stranded oligonucleotide. The samples were place in a Perkin-Elmer ABI Prism 7700... 

Thermal Denaturation Profiles. In the absence of a complimentary target, the molecular beacon expressed approximately 40% fluorescence (relative percentage) at 80°C (Figure 2), indicating that the stem structure had been denatured and the beacons had assumed a coiled conformation. As the temperature was decreased, stable hybridization of the stems was noted at 50°C, when the fluorescence had decreased to 0%. The melting temperature of the stem was determined to be 50-55°C. 

Figure 2. Thermal denaturation profile of molecular beacons.

The cold-denatured state clearly has residual structure in that it is collapsed and exhibits a fairly globular Kratky scattering profile. Truncated, equilibrium unfolded states appear similarly compact (and have been shown by other spectroscopic means to have structure). But what of the highly expanded, urea, GuHCl, or thermally denatured states ... 

Are the irreversible denaturation pathways truly irreversible Thomas and coworkers have demonstrated that the activity of (inactive) oxidized aFGF can be recovered by the addition of the reducing agent dithiothreitol (10). Furthermore, as previously mentioned, various formulation additives have minimized thermally induced aggregation of aFGF. In addition to the physiological implications, these irreversible denaturation pathways also complicate thermodynamic analyses of protein stability, particularly those which rely on van t Hoff analysis of denaturation profiles. 

Thermal stability screens measure the increase in fluorescence intensity of a hydrophobic fluorescent probe that binds to proteins as they unfold during thermal denaturation. Automation of this assay in plate format has allowed rapid screening of libraries for compounds that improve the thermal stability profile of the target protein and has been particularly successful in identifying hits against active site pockets [152,153], and has recently been demonstrated in screening for inhibitors of Bel interactions and human MDM2/p53 [142, 154],... 

As mentioned before, thermal denaturation of short parallel quadruplexes is often irreversible (illustrated in Figure 3(a)). One possibility to circumvent this problem is to record the denaturation profile after an extremely long equilibrium time at each temperature. However, this hysteresis phenomenon is sometimes extreme no renaturation is observed at low strand concentration or unacceptable incubation times are required. Even at a rate as slow as... 

Thermal denaturation studies of DNA and DNA-protein complexes are performed on a UV spectrophotometer equipped with temperature controlled cell holders. Melting of the DNA duplex is indicated by a cooperative hyperchromism at 260 nm. Stabilization by Sac7d is most readily observed with poly[d(AT)]-poly[d(AT)] because of its intrinsic low stability, especially in low salt. The UV melting curve of poly[d(AT)] poly[d(AT)] in 0.01 M K2HPO4 is sharp with a Tm of 43.5° (Fig. 9). In the presence of Sac7d, the melting profile of poly[d(AT)]-poly [d(AT)] broadens and the Tm increases by as much as 33°C for solutions with an excess of Sac7d. The observed Tm for the complex depends on the concentration of protein. 

B. Kessler, Interactions in vitro between gibberellins and DNA by optical rotatory profile of the thermal denaturation of DNA-gibberellin complexes, Biochim. Biophys. Acta 232, 611-613(1971). 

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