Point isodichroic

As a prelude to our binding studies, the secondary structure of aPNA itself was examined using CD spectroscopy [52]. The first aPNA to be studied was the tail-to-tail bl dimer, [Ac-Cys-Gly-Ser -Asp-Ala-Glu-Ser -Ala-Ala-Lys-Ser -Ala-Ala-Glu-Ser -Ala-Aib-Ala-Ser -Lys-Gly-NH2]2- The far-UV CD spectra of this aPNA in water at 30 °C showed the double minimum at 220 nm (n-n transition) and 206 nm (n-n transition) as well as the maximum at 193 nm (n-n transition), characteristic of a peptide a-hehx. Upon increasing the temperature, the intensity of the minimum at 200 nm decreased indicating a transition from a-helix to random stracture. An isodichroic point at 202 nm was suggestive of a temperature-depen-dent a-helix to random coil transition. The helical content of this T5(bl)-dimer at 20°C in water was estimated to be 26% [40]. 

The fraction of Pn conformer in denatured proteins can be roughly estimated from the work of Park et al. (1997). They analyzed the data of Drake et al. (1988) on poly(Lys) over the temperature range of —100 to +80°C plus their own data on the peptide AcYEAAAKEAPAKEAAAKANMHs at temperatures from 0° to 90°C. They used a two-state model, justified by the tight isodichroic points observed in each system, and derived limiting 222 nm ellipticity values of +9500 deg cm2 dmol-1 for the Pn conformation and —5560 deg cm2 dmol-1 for the high-temperature ensemble of conformers. This leads to Eq. (1) (Bienkiewicz etal., 2000) ... 

If the helix and unordered forms are the only forms present, as indicated by an isodichroic point, the fraction of helix, fa is given by ... 

In Section 7.7.3.3, methods for quantitating a-helix and other secondary structural types in peptides were described. These are generally applicable to a series of peptides in which a regular conformation [a-helix, (3-sheet, poly(Pro)II] is in equilibrium with an ensemble of unordered conformations, as evidenced by an isodichroic point observed over a range of temperature, pH, or solvent composition. 

A two-state transition is usually identified by all spectroscopic probes changing simultaneously as the equilibrium changes. The far ultraviolet circular dichroism signals, which are a measure of secondary structure, should change in parallel with the near ultraviolet, which are a measure of tertiary structure. Fluorescence and near ultraviolet absorbance spectra also probe tertiary structure and should change in parallel with each other and the circular dichroism spectra. Ideally, there should be isosbestic or isodichroic points where spectra converge. 

Fig. 3.—pH Dependence of the Circular Dichroism Spectra of D-Glycosiduronic Acids, Illustrated for Methyl a-D-Mannopyranosiduronic Acid. [The isodichroic point at 226 nm shows the existence of two species (carboxylic acid and carboxylate) in simple equilibrium. From Ref. 85 published here by permission.]... 

The evolution of the circular dichroism curves during the neutralization of the pectinic acids by sodium and calcium hydroxides were therefore followed in order to obtain more informations on the conformational changes. The neutralization of sample 5 (a pectin with a DE of 40% with a statistical distribution of the free carboxyl groups), either with sodium or calcium hydroxides leads to a regular decrease both in X and in differential extinction coefficient (A e) which progressively reach values for pure sodium and calcium forms, respectively. This is entirely consistent with a simple ionization process and all the curves pass through the isodichroic point at about 200 nm. 

From these results it is not possible to conclude definitively on the number of conformational states which exist depending on the ionic form. The results suggest that three conformations exist, one in acidic form, perhaps corresponding to a helix with a three fold symetry, one in sodium form and another in calcium form which may be reasonably described by a helix with a two fold symetry. In an other hand, only one isodichroic point was observed and this result may rule out the possibility of three conformations. The literature is also contradictory (24-26) in this respect and further work must be carried out. 

The CD and absorption spectra both fell into two families of spectra, as shown by two sets of isodichroic and isoabsorptive points. Spectra of seimples containing 68% or more of d(CT)]2 had isodichroic points at 269 and 231 nm and isoabsorptive points at 258 and 238 nm. CD spectra of samples with 68% or less of d(CT)i2 showed an isodichroic point at 240 nm, and intersections of the spectra clustered at 273-275 nm. Absorption spectra of these samples had isoabsorptive points close to 268 and 230 nm. Slight variations of 1-2 nm in the wavelengths of equal CD or... 

CD has been used extensively to follow the equilibrium between helix and unordered conformation. Peptides undergoing helix-coil transitions typically exhibit an isodichroic point near 203 nm as the temperature, solvent, pH, or ionic strength is varied. The observation of an isodichroic... 

Observing the transition with a different technique (e.g. calorimetry, NMR, or circular dichroism) can test the two-state assumption. The observation of isosbestic or isodichroic points in the absorbance or circular dichroism spectra, respectively, is diagnostic of a two-state transition (6). Alternatively, the melting profile can be monitored at a different wavelength, so that contributions from different nucleotides are emphasized. Deriving thermodynamics from the concentration dependence of the Tm (see below) can also test the two-state assumption. If all methods give the same results, the two-state approximation is validated since the methods have different sensitivities to each species. 

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