Intensity Of Circular Dichroism Bands Research Articles

Comparative analyses of helical properties in asymmetric 1,2-diacyl-sn-glycerols by means of circular dichroism and proton NMR spectroscopies: notable effects of substituting groups at sn-3 position

The titled 1,2-diacyl-sn-glycerol constitutes a common asymmetric backbone of cell-membrane phospholipids and glycolipids. The acyclic sn-glycerol skeleton allows flexible conformational changes by free rotation along each of the glycerol sn-1,2 and sn-2,3 C–C single bonds in solution. It is generally believed, however, that the sn-1,2 tail position adopts two gauche conformers [gt-(+) and gg-(−)] near exclusively to display an ordered conformation. In the present study, our interest was focused on the fact that the two gauche conformers possess helical signs which are the reverse of each other. We examined (+)- and (−)-chirality arising from the disparity between the two helical conformers using a series of 1,2-dibenzoyl-sn-glycerols 2–8 as model compounds, which possess 1,2-dibenzoate chromophore for circular dichroism analysis, while having variable substituent groups at the sn-3 position. First, the analysis was conducted with the circular dichroism ‘dibenzoate exciton coupling methodology’ of Nakanishi and Harada followed by 1H NMR analysis to calculate the time-averaged populations (%) of the three staggered conformers, that is, gt-(+), gg-(−) and tg. The results are discussed in terms of the ‘helicity index’ which covers information on the sign and intensity of circular dichroism bands in circular dichroism analysis as well as on the helical disparity (%) and the total volume (%) of the two helical conformers in 1H NMR analysis. The two independent spectroscopic approaches were well matched in the estimation of the helical index, which in turn allowed us to conclude that the sn-3 substituting groups have diverse effects on the helical properties of 1,2-diacyl-sn-glycerols in solution.

Hydrogen‐Bond Lengths in Polypeptide Helices: No Evidence for Short Hydrogen Bonds

The long and the short of it: N⋅⋅⋅O distances less than 2.8 Å (see picture) have been implicated in determining the 220 nm circular dichroism band intensity in helical peptides. An analysis of helical peptide crystal structures, however, reveals the average experimentally determined hydrogen-bond lengths are 2.978–3.113 Å (α helix) and 2.907–3.211 Å (310 helix).

Interactions of an Electron-Rich Tetracationic Tentacle Porphyrin with Calf Thymus DNA

The interactions between the water-soluble tentacle porphyrin meso-tetrakis[4-[(3-(trimethylammonio)propyl)oxy]phenyl]porphine (TBOPP) and calf thymus (CT) DNA had been found to exhibit unusual features: (a) At pH 7.0, TBOPP underwent extensive self-stacking along the DNA surface (Marzilli, et al. J . Am. Chem. SOC. 1992, 114, 7575); (b) the DNA-bound form of TBOPP was extensively protonated even at pH 7.0 (Petho, et al. J . Chem. Soc., Chem. Commun. 1993, 1547). In the studies presented here, the formation of the TBOPP-DNA adduct was investigated under various conditions of pH, salt concentration, [porphyrin]/[DNA base pair] ratio (R) , and absence/presence of buffer. Under all conditions studied, TBOPP was an outside binder. Methods employed included circular dichroism (CD), UV-visible absorbance, and fluorescence spectrophotometry. Timedependence experiments were also conducted in order to detect any change in binding characteristics over time. Evidence for outside-bound, self-stacked forms of TBOPP included a large conservative induced visible CD feature, whereas the evidence for DNA-bound, protonated TBOPP included a Soret band red-shifted to 451 nm, a single positive induced CD band at 45 1 nm, and a red-shifted fluorescence band at 740 nm. At least two forms of the outside-bound, self-stacked porphyrin were evident. At high R (0.25), hypochromicity of the Soret band and a conservative type CD feature indicated that TBOPP was extensively stacked. At lower R (0.05), increases in the Soret band intensity and changes in the band shape suggested a less extensively stacked form, but this form of TBOPP had a larger conservative feature in the CD spectrum. The intensity of the conservative band suggested that the less stacked form is very stable at R = 0.05. The CD data are consistent with a model in which the interacting TBOPP in the less stacked form has a different relative orientation than the more stacked form. Both high salt concentration and PIPES buffer appeared to stabilize the outside-bound, self-stacked form of TBOPP. At R = 0.01, a decrease in CD band intensity suggested that the porphyrin was less stacked than at R = 0.05; this change in stacking occurred over 1-2 h. Under these low R conditions where outside-bound self-stacking is less favored, the degree of TBOPP protonation is highest. The conservative-type CD spectrum changed to a simple positive CD band upon protonation of TBOPP, indicating that the binding mode underwent a transition from outside binding with self-stacking to simple outside binding. High salt concentration (100 mh4 NaCl) was found to decrease the degree of protonation, and the presence of PIPES buffer also decreased the degree of protonation at pH 7.0. Under conditions which favored protonation (low salt or no PIPES), the degree of protonation was found to increase gradually during the first few hours after sample preparation. Since in the time course of the experiment the total contents of the solution were unchanged, and since the isolated unstacked porphyrin should protonate quickly, these results suggest that stacking inhibits protonation of the DNA-bound porphyrin.

Protein-dependent conformational behavior of DNA in chromatin.

Information from circular dichroism (CD) and DNA thermal denaturation has been used in concert to study the conformational behavior of DNA in the extended 11-nm fiber of chromatin isolated from HeLa nuclei. The histone-dependent conformational states of the system were investigated by selectively removing the hydrophilic histone domains with trypsin. These were compared to acetylated chromatin from the same source. The integrated intensity of the positive CD band for DNA above 260 nm is found to increase with the content of relatively unstressed B-form DNA. This same increase is observed along the series of whole, H1-stripped, and trypsinized chromatin samples as protein is removed. Hence, the ratio of percent hyperchromicity to integrated CD band intensity of the respective melting transitions provides useful information on the conformational state of DNA in the three principal regions of the chromatin fiber: the central loop and flanking nucleosomal regions and the linker. Results from this study suggest that central loop DNA in both hyperacetylated and control chromatin relaxes as protein is removed. However, hyperacetylated chromatin shows significantly less dependence than control chromatin upon core histone hydrophilic domains in the flanking and linker regions. Thus, histone hyperacetylation evidently relaxes DNA in chromatin with no major overall conformational changes. A possible role of histone hyperacetylation may therefore be to reduce cooperativity in the unfolding transition in chromatin and thus provide for greater localized control of unfolding during transcription.

Formation of Chiral Aggregates of Acylamino Acids in Solutions

Abstract An alcoholic solution of optically active acylamino acid (AAA) was found to show a circular dichroism (CD) band at 212 nm. The CD band should be inherent to the solution system, since AAA and alcohol per se have no absorption band at this wavelength. The CD band intensity decreased on addition of urea and rise in temperature. This is attributed to the formation of chiral aggregate through hydrogen bonds among AAA molecules. Hydrophobic interaction seems to be of minor importance for the formation of the chiral aggregate in alcoholic solution, since the variation of the chain length in the acyl group or addition of myristic acid did not affect the magnitude of profile of the CD band. In aqueous solutions, salts of optically active AAA formed micelles with hydrophobic interactions among the molecules, and only in the case of optically active monosodium salt of N-acylglutamic acid (AGS) showed a CD band at 220 nm resulting from the formation of chiral hydrogen bonds at concentrations above the critical micelle concentration (CMC). This type of chiral aggregation may be caused by the existence of free carboxyl group in the optically active AGS molecule.

Fluorescence and circular dichroism studies on human serum low density lipoprotein particles and lipid-depleted derivatives.

AbstractThe low density liporpotein from human serum, and derivitives prepared free of neutral lipids and total lipids, have been studied by fluorescence and circular dichorism methods. Removal of the neutral lipids had little effect on the tryptophan fluorescence at neutral pH. However, by the criteria of circular dichroism, over the range of 200 nm to 250 nm, there was a reduction in secondary structure of over 75%. Removal of the remaining phospholipids resulted in a qualitatively different structure by both fluorescence and circular dichroism criteria.Neutral lipids were removed from LDL in a step‐wise fashion in order to determine the exact amount of neutral lipid required for the native circular dichroism spectrum. The circular dichroism band intensity was constant until approximately 10% of the total cholesterol (as cholesterol ester) remained. The intensity then abruptly dropped as more cholesterol was removed.We concluded that the two spectroscopic methods report on two distinct aspects of LDL structure. The tryptophan fluorescence appears to be sensitive to the presence of phospholipids. The circular dichroism, however, appears to be sensitive to the binding of a small amount of neutral lipid. These findings suggest that a functional and geometric separation of binding sites may exist for these two classes of lipids. Such a distinction is predicted by the icosohedral model of the quaternary structure of LDL. In this model, the phospholipids are located on the surface of the particle, in the holes of an icosohedrally symmetric surface network of protein subunits; the neutral lipids are located in the particle core.Finally, we suggest that functional significance may be attached to our finding that relatively few cholesterol ester molecles are needed to maintain the native secondary structure of LDL. This provides a mechanism whereby the amount of bound neutral lipid could be raised or lowered (for transport and transfer to cells) without affecting the protein in any structurally significant manner.

Effects of Denaturing Agents on the Phenylalanyl Circular Dichroism Bands of Horseradish Peroxidase Isoenzymes and Apoisoenzymes

Abstract The influence of protein conformation upon the circular dichroism (CD) bands of two peroxidase isoenzymes (A1 and C) was investigated by using guanidine hydrochloride and other denaturing agents. In all four proteins, guanidine hydrochloride markedly reduces the 221-nm CD band, indicating disruption of the ordered peptide backbone conformation. Similarly guanidine hydrochloride greatly diminishes the near ultraviolet CD bands of the tyrosyl and tryptophanyl side chains, which implies that their orientation has been disrupted. In contrast, guanidine hydrochloride treatment of peroxidase C brings out phenylalanyl CD fine structure at 268 and 261 nm, even though none can be detected in the native conformation. The intensities of these CD bands in the denatured peroxidase C are comparable to those of an equivalent concentration of unoriented phenylalanyl model compound. These findings indicate that in the native peroxidase C many of the phenylalanyl side chains have fixed orientations producing unique interactions with the surroundings. Apparently the rotatory strength of each oriented phenylalanyl side chain may be weak or strong and either positive or negative. The native peroxidase C may have as many as 23 oriented phenylalanine residues; summing the individual contributions leads to nearly complete cancellation of CD intensity. In the denatured peroxidase C, even though the unoriented phenylalanyl side chains have weak CD bands, all have nearly the same CD spectrum, thereby precluding cancellation among the residues. Thus the observed intensity of the phenylalanyl CD bands increases when the orientations of the phenylalanyl side chains are disrupted in peroxidase C. Guanidine hydrochloride and other denaturing agents do not cause any detectable change (l20%) in the intensity of the 268- and 261-nm phenylalanyl CD fine structure observed in the native peroxidase A1 or either of the apoisoenzymes. Other evidence suggests that, at least for peroxidase A1, the observed intensities of the phenylalanyl CD bands may remain constant even though the local environment is altered.