High-resolution circular dichroism of N-acetyl-L-phenylalaninamide: solvent effects

Biological membranes consist mainly of lipids and proteins. At present, the structure of the lipid phase appears to be established, but hypotheses on the molecular organization of the protein are difficult to support. Thus the deformation behavior of whole human erythrocyte ghosts, ghosts after the selective removal of lipids and ghosts stripped of lipids as well as nonlipid components have been examined in the hope of securing indirect information on the organization of the protein. It has been found that large localized deformations result in partial membrane failure and long uniformly wide fibrils, frequently in excess of 3000 Å, are drawn across the rupture. These data are interpreted in terms of currently favored membrane models and the possibility of a fibrous membrane framework consisting predominantly of protein is reviewed. The behavior of the membrane in its various stages of extraction is compared and contrasted to that of synthetic polymer films of known organization.     

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.     

Capillary circular dichroism

Circular dichroism (CD) has become an increasingly important tool in the study of biological molecules as it enables structural information to be obtained nondestructively on solution-phase samples. However, sample requirements for CD are often seen as being too high with protein backbone measurements in standard cuvettes typically requiring ～100-300 μL of 0.1 mg/ml protein. To address this issue, we have designed a new form of CD sample holder, which reduces the sample requirements of the technique by two orders of magnitude, with a sample requirement of less than 3 μl. This sample saving has been achieved through the use of extruded quartz capillaries, the sample being held within the internal diameter of the quartz capillary through capillary action. The extruded quartz capillaries exhibit remarkably little birefringence, although still transmitting high energy UV circularly polarized light. The optics associated with capillaries were investigated. A configuration has been adopted with the light beam of the spectrophotometer being focused in front of the front face of the capillary using a biconvex lens and advantage being taken of the additional focusing effect of the capillary itself. The focusing is vital to the low wavelength performance of the cell, where we have acquired reliable data down to 180 nm using a Jasco J-815 spectrophotometer. The system performance was validated with Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid), concanavalin A, lysozyme, and progesterone.     

The differential scattering of circularly polarized light by chloroplasts and evaluation of their true circular dichroism

Chloroplasts isolated from pea leaves display an intense circular dichroism in the range 600 to 720nm. Circularly polarized light is also differentially scattered by chloroplasts, and this effect can be confused with circular dichroism. By using an instrumental modification it was possible to distinguish, and record separately, the ellipticities of the transmitted light (circular dichroism) and of the scattered light in the same c.d. instrument. By means of a light-scattering apparatus, the intensity of unpolarized light scattered by chloroplasts was measured as a function of wavelength and of angle. This measurement allowed the aforementioned ellipticities to be corrected for mutual interference. At a concentration of 4mug of chlorophyll/ml (the optimum practical concentration of chloroplasts at which there was no significant interaction of scattering and absorption effects) spectra of true circular dichroism (circular differential absorption) and circular differential scattering were obtained. The former showed maxima, positive at 688nm and negative at 676nm, with an intensity Deltatheta=8.3m degrees .litre.(mg of chlorophyll)(-1).cm(-1). The latter had a maximum at 683nm with an intensity of +47m degrees with respect to the solvent baseline; this value is independent of the concentration of chloroplasts in dilute suspensions. It is suggested that the intense circular dichroism of chloroplasts reflects specific chlorophyll-chlorophyll interactions in the light-harvesting pigment. The advantages of this method for determining the c.d. of scattering suspensions over those of other investigators are discussed.     

Vacuum ultraviolet circular dichroism of fibronectin. Dominant tyrosine effects

The spectroscopic authenticity of a very intense negative band at about 183 nm reported previously from conventional circular dichroism (c.d.) studies of bovine plasma fibronectin has now been confirmed by vacuum ultraviolet c.d. measurements on two prototype spectrometers, one using a conventional light source and the other using synchrotron radiation. Closely similar spectra were obtained from both instruments, and from both solid films and solutions. The spectra show no obvious parentage in the known c.d. of the peptide backbone, but have marked similarities to the c.d. of N-acetyltyrosineamide, both in the strong band at 183 nm and in a characteristic positive band at 230 nm, It is concluded that the c.d. of fibronectin is dominated by contributions from tyrosine side-chains and that, as suggested previously, these may provide a sensitive probe for molecular organization and interactions.     

Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data

The effects of spectral magnitude on the calculated secondary structures derived from circular dichroism (CD) spectra were examined for a number of the most commonly used algorithms and reference databases. Proteins with different secondary structures, ranging from mostly helical to mostly beta-sheet, but which were not components of existing reference databases, were used as test systems. These proteins had known crystal structures, so it was possible to ascertain the effects of magnitude on both the accuracy of determining the secondary structure and the goodness-of-fit of the calculated structures to the experimental data. It was found that most algorithms are highly sensitive to spectral magnitude, and that the goodness-of-fit parameter may be a useful tool in assessing the correct scaling of the data. This means that parameters that affect magnitude, including calibration of the instrument, the spectral cell pathlength, and the protein concentration, must be accurately determined to obtain correct secondary structural analyses of proteins from CD data using empirical methods.     

A circular dichroism microspectrophotometer

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.     

Linear dichroism and circular dichroism in photosynthesis research

The efficiency of photosynthetic light energy conversion depends largely on the molecular architecture of the photosynthetic membranes. Linear- and circular-dichroism (LD and CD) studies have contributed significantly to our knowledge of the molecular organization of pigment systems at different levels of complexity, in pigment–protein complexes, supercomplexes, and their macroassemblies, as well as in entire membranes and membrane systems. Many examples show that LD and CD data are in good agreement with structural data; hence, these spectroscopic tools serve as the basis for linking the structure of photosynthetic pigment–protein complexes to steady-state and time-resolved spectroscopy. They are also indispensable for identifying conformations and interactions in native environments, and for monitoring reorganizations during photosynthetic functions, and are important in characterizing reconstituted and artificially constructed systems. This educational review explains, in simple terms, the basic physical principles, and theory and practice of LD and CD spectroscopies and of some related quantities in the areas of differential polarization spectroscopy and microscopy.     

The effects of pH and temperature on the circular dichroism of human erythrocyte membranes.

The effects of pH and temperature on the structure of human erythrocyte membranes were studied by circular dichroism (CD). The results obtained demonstrate that the membrane CD spectra undergo significant changes when the pH of the solution deviates from its native pH range of 7 to 8. Spectral changes in the acidic pH region include drastic reductions and slight shifts in the CD signal which may reflect a decrease in alpha-helical content of the proteins and/or an increase in optical artifacts, both of which are irreversible. In the alkaline pH region, dramatic increases in ellipticity and blue-shifts in the spectra are observed between pH 8 and 10. In addition, the spectra more closely resemble those reported for membrane samples where the spectral distortions have been removed. The changes in the alkaline region are demonstrated to be only partially reversible and may be due to conformational alterations in the membrane proteins and/or to a reduction in optical distortions. Thermal stability studies reinforce the irreversible behavior of the membrane samples.     

Heparin-induced circular dichroism of chloroquine

Circular dichroism (CD) and UV/Visible absorption (UV/Vis) spectroscopy techniques were used to investigate the interaction between heparin and chloroquine, an antimalarial drug that has shown potential as an anti-prion agent. CD spectra of rac-chloroquine upon addition of heparin provide evidence of glycosaminoglycan (GAG) binding, support recent findings suggesting that interactions between heparin and antimalarial drugs are largely due to electrostatic interactions, and represent the first reported GAG-induced CD signal of a bicyclic, aromatic compound. The association constant (∼10³ M⁻¹) between chloroquine and heparin was calculated from a UV titration curve and provided additional insight into the nature of the association between these two compounds.