Fourier transform infrared spectroscopy indicates a major conformational rearrangement in the activation of rhodopsin.

The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.

An A-form of poly[d(A-C)].poly[d(G-T)] induced by mercury (II) as studied by UV and FTIR spectroscopies.

The conformational changes of poly[d(A-C)].poly[d(G-T)] induced by Hg(ClO4)2 in aqueous solution have been studied using UV absorption and fourth derivative spectrophotometries, and FTIR spectroscopy. The UV absorption and fourth derivative spectra reflect changes in the polynucleotide stacking interactions as a result of the metal-polynucleotide interaction. The fourth derivative spectra do not indicate a Z-form either at low or at high metal-to-polynucleotide ratios. Furthermore, the infrared spectrum at high metal-to-polynucleotide ratio (r = 1.2; r = [Hg(ClO4])2/[nucleotide] molar ratio) has the main features of an A-form, in contrast with previous CD studies which proposed that the polynucleotide adopts a Z-form under these conditions. The nature of a different conformation of the polynucleotide induced at low r-ratios (r < or = 0.2) is discussed.

Quantitative characterization of the structure of rhodopsin in disc membrane by means of Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy has been used for the detailed characterization and quantification of the secondary structure of bovine rhodopsin in native disc membranes. FTIR spectra were obtained in aqueous media, both in 1H2O and in 2H2O. Analysis of spectra by means of Fourier self-deconvolution, complemented with maximum likelihood restoration and Fourier derivative, has allowed the characterization of major amide I secondary structure-sensitive component bands of structural relevance which had not been detected before. In consequence, we show a richer secondary structure for rhodopsin than previously described. Our results indicate a total regular helix content around 51%, which would include not only the main alpha 1-type helix but also 3(10)-like helix. The presence of distorted helicoid sequences might furthermore increase to a certain extent the total helix amount. It is also indicated that a significant proportion of the amino acid residues are involved in extended/beta-structures and in reverse turns, as well as in "random" segments, which had not been directly demonstrated before. 61 +/- 4% of rhodopsin is determined to be solvent-accessible, which is a substantially higher value than previously reported. Helices account for most of the inaccessible moiety.

An A-form of poly(amino2dA-dT).poly(amino2dA-dT) induced by polyamines.

The effect of the naturally occurring polyamines spermidine and spermine on poly(amino2-dA-dT).poly(amino2dA-dT) conformation has been studied by UV, CD, and IR spectroscopies. It is shown that a conformational transition is induced in poly(amino2dA-dT).poly(amino2dA-dT) by micromolar concentrations of the polyamines (30 microM) in low-salt aqueous solution. The analysis of our results, in view of previously published studies on conformational properties of the amino polynucleotide, indicates the resulting conformer to be an A-form. Interestingly, the polyamine concentration at the midpoint of the transition is the same in both cases. This provides further evidence that the coordination of positively charged counterions to DNA is determined largely from the DNA structure, probably with an important role for the sequence, and less from the nature of the ions.

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC).

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.

Study of the structure of arrestin (S-antigen) from bovine photoreceptors by FTIR spectroscopy.

Fourier transform-infrared spectroscopy has been used for the study of the secondary structure of arrestin from bovine retina rod cells. Spectra have been obtained in H2O and in D2O media. Resolution enhancement of the amide I secondary structure-sensitive overlapped component bands has been achieved by means of Fourier self-deconvolution and Fourier derivation. In order to obtain a quantitative estimation of the proportion of amino acid residues involved in each type of secondary structure, bands at the resolved frequencies have been curve-fitted to the deconvolved amide I contour by means of a least-squares best-fitting iterative program. The analysis of the results suggests that the secondary structure of arrestin comprises 56-63% of extended strands, 12-19% of turns and bends, 15% of alpha-helices and 10% of undefined and irregular segments.

9-aminoacridine inhibits the B-Z transition of poly(dA-dT).

9-Aminoacridine is the parent compound of a family of pharmacologically active model substances that bind to DNA through intercalation between base pairs. In the present study we show that 9-aminoacridine inhibits the B-to-Z isomerization of poly(dA-dT) in conditions that otherwise cause it to occur (5 M NaCl and 123 mM Ni(ClO4)2). Higher concentrations of Ni(ClO4)2 (155 mM) are able to induce the Z-form due to the disruption of the drug-polynucleotide interaction by the metal ion. Additionally, the dye reverses the Z-form in certain conditions. Thus, the data from this study indicate that 9-aminoacridine binds preferentially to the B-form of poly(dA-dT).

FT-IR spectroscopic study of the poly(amino2dA-dT) duplex in Mg(2+)-containing solution and in films.

The alternative structures of the synthetic poly(amino2dA-dT) duplex have been studied using infrared spectroscopy in films and in solution (D2O and H2O) in the presence and in the absence of magnesium salt. In solution without magnesium salt, the polynucleotide exists in a B genus conformation with some of the sugar puckers possibly in the C3'-endo/anti geometry. In magnesium-containing solution (66 mM MgCl2), however, we report infrared spectra of Mg(2+)-poly(amino2dA-dT) which have characteristic marker bands of the A form. Film samples in 70% relative humidity (RH) give similar infrared spectra to those of the polynucleotide obtained using Mg2+. Thus, when analyzed in comparison with previously reported infrared spectra of other oligo and polynucleotides, our data show that double helical poly(amino2dA-dT) goes into the same (or very closely related) conformation in dehydrated films as in solutions containing Mg2+.

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study.

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.