Positive Dichroism Research Articles

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT) 8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.

Dynamic infrared linear dichroism study of high-density and low-density polyethylene near the β-transition temperature

High-density polyethylene (HDPE) and low-density polyethylene (LDPE) films under small-amplitude oscillatory strain were examined using time-resolved infrared (IR) linear dichroism spectroscopy. A negative dynamic dichroism at 1473 cm −1 and a positive dynamic dichroism at 1463 cm −1 were observed at +32°C (above the β-transition temperature, T β. This observation is interpreted to mean that above T β the orthorhombic crystallographic b axis reorients parallel to the direction of applied strain, while the crystallographic a axis reorients perpendicular to the strain direction for both HDPE and LDPE. Interestingly, the dynamic dichroism of both bands changes sign at −50°C (below T β), indicating that the dynamic reorientation directions for the crystalline a and b axes are shifted below T β. This result clearly shows that different deformation mechanisms operate above and below T β. Even though the IR peaks in this region are made up of overlapping bands from both crystalline and amorphous components, they are easily resolved in this experiment. The high spectral resolution was achieved because the electric dipole transition moments associated with individual bands reorient in different directions, at different rates, and to different extents under dynamic strain.

Differences in conformations of covalent adducts derived from the binding of 5-and 6-methylchrysene diol epoxide stereoisomers to DNA

The conformations of adducts derived from the covalent binding of four different isomeric diol epoxide derivatives of 5- or 6-methylchrysene to native double-stranded calf thymus DNA were studied by linear dichroism techniques. Out of four isomers investigated here, only the R,S,S,R enantiomer of anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene, (+)-5-MeCDE, is highly tumorigenic and mutagenic toward Salmonella typhimurium TA100; the S,R,R,S enantiomer, (-)-5-MeCDE, and the corresponding R,S,S,R and S,R,R,S enantiomers of anti-1,2,-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene are non-tumorigenic and much less mutagenic than (+)-5-MeCDE. [Melikian et al., (1988) Cancer Res., 48, 1781-1787] Only the DNA adducts derived from the binding of (+)-5-MeCDE are characterized by a pronounced positive linear dichroism signal at 308 nm due to the phenanthrenyl residue which is tilted at an angle of 45-48 degrees with respect to the average orientations of the axes of unoriented DNA segments. The phenanthrenyl residues derived from the covalent binding to DNA of the other three inactive or less active isomers appear to be unoriented. The defined orientation of the covalently bound phenanthrenyl residues derived from (+)-5-MeCDE corresponds to adduct conformations which are similar to those obtained from the binding of the highly tumorigenic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene stereoisomer and other highly active bay-region diol epoxide derivatives to DNA. These findings provide further evidence that there is a correlation between DNA adduct conformation and biological activities for these series of polycyclic aromatic diol epoxide derivatives with R,S,S,R absolute configuration and which are known to bind predominantly to N2 of guanine.

Orientation of the chromophore transition moment in the 4-leaved shape model for pea phytochrome molecule in the red-light absorbing form and its rotation induced by the phototransformation to the far-red-light absorbing form

Orientation of the chromophore transition moment in pea phytochrome (subunit molecular mass, 114 kDa) in the red-light-absorbing form (P r) and its rotation induced by the phototransformation to the far-red-light-absorbing (P fr) form was studied by measuring linear dichroism of oriented phytochrome prepared by a ‘gel squeezing method’. The phytochrome in P r showed a negative linear dichroism. On the contrary, the P fr form showed positive linear dichroism. Assuming that the molecular axis for the two-fold rotational symmetry in the ‘4-leaved shape model’ proposed recently for the dimeric molecular structure of the phytochrome [(1989) FEBS Lett. 247, 139–142], is oriented in parallel with the direction of the external constraint, the average azimuthal angle between the molecular axis and the transition moment of the chromophore was calculated as 59° or 121° for P r and 52° or 128° for P fr. These results explain well the polarotropic responses by phytochrome observed in plant organisms.

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA.

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.

Fundamental Optical Absorption Edge in MnIn2Te4 Single Crystals

The optical properties are studied in the region of the fundamental absorption edge of MnIn2Te4 single crystals (142m, defect stannite) oriented along (100) and (001). The width of the energy gap is measured to be Eg = 1.55, 1.425, and 1.41 eV, for the temperatures 77, 300, and 370 K, respectively. The α(ℏω) spectra are established to follow Urbach's rule throughout the temperature range studied, the steepness of the absorption edge remaining constant. Crystal annealing followed by fast cooling shifts the absorption edge longward by 50 meV, the exponential character of α(ℏω) persisting within the whole range T = 77 to 370 K. An analysis shows the optical absorption in the region of the fundamental edge to be governed by the sum of the effects of the density-of-states tails associated with a high concentration of lattice vacancies and of the electron-phonon interaction. The optical linear dichroism of the absorption edge of MnIn2Te4 crystals with defect stannite structure is discovered and studied. It is found that the crystals belonging to the tetragonal syngony, such as MnIn2Te4 (142m) and CuInSe2, CuInTe2 (142d), and exhibiting a weak tetragonal lattice deformation τ ≈ 0, reveal, in contrast to most chalcopyrites with τ > 0, a positive linear dichroism of transmittance close to Eg. Minimal direct transitions in MnIn2Te4 are allowed in the E ⊥ c polarization, the crystal splitting of the valence band at the Γ point being positive. [Russian Text Ignored].

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1, N 6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1, N 6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1, N 6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(± 6) °. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 ° with the local helix axis. Some possible structures of the DNA-protein complex are discussed.

Structure of the Tet repressor and Tet repressor-operator complexes in solution from electrooptical measurements and hydrodynamic simulations.

The Tet repressor protein and tet operator DNA fragments and their complexes have been analyzed by electrooptical procedures. The protein shows a positive linear dichroism at 280 nm, a negative linear dichroism at 248 nm, and a strong permanent dipole moment of 3.5 X 10(-27) C m, which is independent of the salt concentration within experimental accuracy. Its rotation time constant of 40 ns indicates an elongated structure, which is consistent with a prolate ellipsoid of 100 A for the long axis and 40 A for the short axis. The time constant can also be fitted by a cylinder of length 78 A and diameter 37 A, which is consistent with nuclease protection data reported on repressor-operator complexes, if the cylinder axis is aligned parallel to the DNA axis. Addition of tetracycline induces changes of the limit dichroism but very little change of the rotation time constant. The rotation time constants observed for the operator DNA fragments show some deviations from the values expected from their contour length; however, these deviations remain relatively small. Formation of repressor-operator complexes leads to some increase of the DNA rotation time constants. Simulations by bead models demonstrate that these time constants can be explained without any major change of the hydrodynamic dimension of the components. The data for the complexes are fitted by bead models with smooth bending of the DNA corresponding to a radius of curvature of 500 A, but at the given accuracy, we cannot rule out that the DNA in the complex remains straight or is bent to a smaller radius of approximately 400 A. Thus, binding of the Tet repressor, which is a helix-turn-helix protein as judged from its sequence, to its operator seems to induce minor bending but does not induce strong bending of the DNA double helix.

The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.

Comparative Study of the Condensation of Chicken Erythrocyte and Calf Thymus Chromatins by Di- and Multivalent Cations

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.

Orientation of the pigments in Photosystem II: low-temperature linear-dichroism study of a core particle and of its chlorophyll-protein subunits isolated from Synechococcus sp.

The orientation of the pigments in the Photosystem II core particle isolated from the thermophilic cyanobacterium Synechococcus sp. has been investigated by linear dichroism spectroscopy at 10 K of macroscopically oriented samples. The absorbance (A), linear dichroism (LD) and LD/A spectra are remarkably similar to those previously reported for a core complex isolated from Chlamydomonas reinhardtii (Biochim. Biophys. Acta 850 (1986) 156–161). The spectra of the Synechococcus core particle are compared to the corresponding spectra obtained on its two main constituent chlorophyll-protein complexes CP2-b (photochemically active) and CP2-c (photochemically inactive). The various features seen in the spectra of the core particle appear well segregated into the spectra of one or the other of the two subparticles without significant loss of orientation of the pigments. The orientation of the chlorophyll macrocycles, with the Y and X optical axis preferentially parallel and perpendicular to the plane of largest cross-section of the particle, respectively, is very similar in the two subparticles. CP2-b contains mainly the beta-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane, while CP2-c contains the beta-carotene pool absorbing around 495 and 465 nm and oriented closer to the normal to the membrane plane. A shoulder at 682 nm in the absorbance and linear dichroism spectra of the core complex is fully segregated in the spectra of CP2-c, thus excluding the possibility that this spectral feature could be assigned to the primary donor of PS II. A negative linear dichroism component peaking around 691 nm (LD 691) in the core particle is mainly segregated in CP2-b together with the photoactive pheophytin acceptor molecule responsible for the 544 nm positive linear dichroism signal (LD 544). While the ratio of the amplitudes LD 691/LD 544 is approximately the same for the core particle and for the CP2-b complex, the amplitude of LD 691 is significantly reduced in CP2-b compared to the core particle.

Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2

Linear-dichroism spectra of Anacystis nidulans at 77 K were determined for whole cells chemically fixed in light State 1 and light State 2. Whole cells were oriented by the squeezed gel technique using 5% gelatin 2.2 M sucrose gels. Peaks with positive dichroism were observed at 638 nm and 688 nm with shoulders at approx. 650 nm and 700 nm. The amplitude of the 650 nm shoulder was greater for cells in State 2 than those in State 1, and the State-2-minus-State 1 difference spectrum had a single peak at 656 nm. The linear dichroism spectrum of phycobilisomes isolated from A. nidulans showed peaks at 635 nm (phycocyanin) and 656 nm (allophycocyanin). The spectrum for thylakoid membranes free of phycobilisomes had one peak at 685 nm with a shoulder at 698 nm. We suggest that the change in dichroism at 656 nm between cells in State 1 and State 2 results from a change in orientation of the allophycocyanin core of the phycobilisome. This result is discussed in the context of our model for the light-state transition in phycobilisome-containing organisms.

Interactions of nucleic acid double helices induced by electric field pulses.

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 X 10(10) M-1 helices s-1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants kd decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(kd) and E2 suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing salt concentration (measured in the range 1-5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 microseconds, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.

The role of histone H1 and non-structured domains of core histones in maintaining the orientation of nucleosomes within the chromatin fiber

Calf thymus chromatin was digested with trypsin and the structural alterations which occured were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.

On the structure of active chromatin: A flow linear dichroism study of chromatin fractionated by nuclease digestion

Nuclei from Ehrlich ascites cells were treated with micrococcal nuclease or DNase I and extracted with 1 mM EDTA. The chromatin fraction released by this procedure showed positive flow linear dichroism (LD) at low salt (2 mM NaCl) while the non-released fraction had negative LD. Furthermore, the chromatin structure responsible for the positive LD was found to be labile: The LD was reduced by heat (37°C) or RNase treatment and inverted to a negative LD by electric fields (10 kV/cm) and by the presence of DNA binding dyes.

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen.

A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dyes is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5-6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.

Synthesis of 1,2-diamino imidazole derivatives by the reaction of benzaldehyde guanylhydrazone with ?-haloalkyl aryl ketones

2-Amino-1-benzylideneamino-4-arylimidazoles were obtained by the reaction of benzaldehyde guanylhydrazone with 4-alkyl- and 4-aryl-Ω-haloacetophenones. Side products of this reaction were cis- and trans-1,1′-bis(benzylideneamino)-4,4′-diaryl-2,2′-azoimidazoles. The corresponding 2-amino-1-benzylideneaminoimidazole and 1-benzyl-ideneaminoimidazo[l,2-a]imidazole were obtained in the reaction of α-bromopropiophenone with benzaldehyde guanylhydrazone. The 2-amino-1-benzylideneamino-4-arylimidazoles were converted by successive reactions to 1,2-diatnino-4-arylimidazoles and then to imidazo[1,2-b]-1,2,4-triazine derivatives —dyes for liquid crystals with positive dichroism.

Chromatin structure studied by linear dichroism at different salt concentrations.

AbstractWe have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease‐digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease‐digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl2. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na+, this can be explained as a screening effect due to the bulk concentration of Na+. With Mg2+ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.

Influence of ionic strength on the dichroism properties of polynucleosomal fibers.

AbstractWe report electric‐dichroism and electron‐microscopic studies of chromatin fibers fixed by protein–protein crosslinking at salt concentrations ranging from 10 to 100 mM. The results confirm a progressive disorganization of the fiber as the salt concentration is lowered. The positive dichroism and large polarizability anisotropy characteristic of the 300‐Å diameter fiber found in 100 mM salt are replaced by negative dichroism and smaller effective polarizability anisotropy or dipole moment for samples fixed at lower salt concentration. We interpret the results in terms of segmental, field‐induced orientation of the disorganized structure which is present in low salt concentrations. We also observed a field‐induced absorbance decrease in chromatin fibers fixed at salt concentration at and below 100 mM. All three optical effects, namely overall orientation of the high‐salt fixed fiber, segmental orientation of the low‐salt fixed fiber, and field‐induced absorbance decrease, occur on roughly the same time scale, 20–100 μs for 50 nucleosome polynucleosomes. The polarizability anisotropy of fibers fixed in 100 mM salt was found to be proportional to the length of the fragment and to the reciprocal square root of the conductivity of the solution used for electric‐dichroism measurements. Addition of Mg2+ to the measurement buffer affected the dichroism amplitude of samples fixed below 100 mM salt but not those fixed at 100 mM salt. The results reinforce the need for caution in interpreting electric‐dichroism measurements on chromatin fibers because of possible field‐induced distortion effects.

Orientation of rhodopsin alpha-helices in in retinal rod outer segment membranes studied by infrared linear dichroism.

Frog retinal rod outer segments, oriented by a magentic field, were shown to contain rhodopsin alpha-helical segments preferentially aligned perpendicular to the plane of the disc membrane, by the technique of infrared linear dichroism. Infrared absorption parallel and perpendicular to the rod axes by peptide C parallel to O groups, whose absorption band contains alpha-helical and random coil components at slightly different frequencies, showed positive dichroism centered on the alpha-helix frequence. We conclude that the alpha-helical portion of the protein has an average orientation in the transmembrane direction. Furthermore, infrared spectra of rods in 2H2O Ringer's solution exhibit two distinct peptide amino group absorption bands: the unexchanged N-2H band, which is nondichroic. This implies that the oriented part of the protein is in the lipid bilayer, supporting a model for rhodopsin with a hydrophobic core containing partially oriented alpha-helices and hydrophilic ends consisting of unoriented polypeptide.