Theoretical optical properties of poly-L-proline

A non-perturbational technique is used to calculate the circular dichroism and absorption spectra of polypeptide chains having conformations similar to that of poly-L -proline II. The method employs a Bogoliubov exciton formalism, from which the various optical terms associated with parallel and perpendicular components of the exciton band are obtained. A simple model for the peptide unit, consisting of three Gaussian absorption bands, leads to reasonable results for the polymer spectra, provided the lowest energy peptide π → π* transition is taken at 207 mμ and the value of the Ramachandran angle Ψ is taken to be 390°. The calculations suggest that the polymer circular dichroism spectrum is the resultant of strong interference among the two Gaussian exciton terms and the non-Gaussian helix term. Consequently, the CD spectrum is very sensitive to the value of Ψ. It is found that the small positive CD band in the vicinity of 230 mμ arises partly from the effect of the static (crystal) field interactions on the n → π* CD band.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

R-phycoerythrins having two conformations for the same aggregate

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.     

Circular dichroism and conformational properties of modeccin,a toxic protein

According to its circular dichroism (CD) spectrum, modeccin, a toxic lectin from the roots of the South African plantModecca digitata, is structurally similar to the ricins and abrins. In nearly neutral and weakly alkaline solutions (pH 7.6–9.0) the CD spectra of modeccin displayed a positive CD band at 190–195 nm and a negative band at 210–220 nm, indicating the presence of some α-helix and β-sheet structures. In the near-ultraviolet zone, we observed positive CD bands at 232 and 245 nm and weak negative bands at 285 and 293 nm. In more strongly alkaline solutions of pH 9.5–10.2 the CD bands in the farultraviolet zone were not affected, but the CD band at 232 nm diminished and the CD band at 245 nm was enhanced. These transitions were reversible. At pH 11.2–11.5 the CD band at 232 nm disappeared completely, and the CD bands in the far-ultraviolet diminished. The CD bands at 285 and 293 nm were affected very little by the alkali, and these bands were assigned to buried tryptophan side chains. Sodium dodecyl sulfate and 2,2,2-trifluoroethanol disorganized the tertiary structure of modeccin and reconstructed the secondary structure into a new form with a higher helix content than in the native protein.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

A non-perturbation method for the optical properties of helical polymers

The optical absorption and circular dichroism (CD) of poly-L -alanine in an α-helical conformation are computed in terms of monomer properties by use of Bogoliubov exciton theory (a non-perturbation method). It was shown earlier that a proper formulation of the theory of CD for large molecular systems permits the use of periodic boundary conditions, a feature which greatly simplifies calculations as well as conceptual understanding. If is found that the shape of the observed CD spectrum can be explained in terms of the two rotational strength components and an electric dipole strength component referred to as the helix term. Comparisons of the results of perturbation theory and Bogoliubov exciton theory show that perturbation theory is valid for absorption but is inadequate for CD if the coupling among monomers is sufficiently large lo predict the observed hypochromism.     

Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films.

The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

Circular dichroism studies of α-aminoisobutyric acid-containing peptides: Chain length and solvent effects in alternating Aib-L-Ala and Aib-L-Val sequences

The CD spectra of the peptides Boc-X-(Aib-X)_n-OMe (n = 1, 2, 3) and Boc-(Aib-X)₅-OMe, where X = L -Ala or L -Val have been examined in several solvents. The X = Ala and Val peptides behave similarly in all solvents, suggesting that the Aib residues dominate the folding preferences of these peptides. The decapeptides adopt helical conformations in methanol and trifluoroethanol, with characteristic negative CD bands at 222 and 205 nm. In the heptapeptides, similar spectra with reduced intensities are observed. Comparison with nmr studies suggest that estimates of helical content in oligopeptides by CD methods may lead to erroneous conclusions. The pentapeptides yield solvent-dependent spectra indicative of conformational perturbations. Peptide association in dioxane results in an unusual spectrum with a single negative band at 210 nm for the decapeptides. Disaggregation is induced by the addition of methanol or water to dioxane solutions. Aggregation of the heptapeptides is less pronounced in dioxane, suggesting that a critical helix length may be necessary to promote association stabilized by helix dipole–dipole interactions.     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

Individual tyrosine side-chain contributions to circular dichroism of ribonuclease

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.     

Random copolymers of alpha-benzyl-L-aspartate with ortho- and para-nitrobenzyl-L-aspartates. Intrinsic and extrinsic dichroic bands and the left-to-right-handed alpha-helical transition.

Copolymers of benzylaspartate with the corresponding para-nitro and ortho-nitro derivatives are known to undergo, at increasing nitro content, a transition from a left-handed α-helical conformation to a right-handed α-helical conformation. The circular dichroism associated with the n, π* transition of the amide group, as well as the extrinsic bands associated with the nitrobenzyl chromophore, have been studied for these copolymers as a function of the nitro content. The circular dichroism associated with the 330-nm extrinsic band is shown to present an abrupt change, which parallels the change in the 222-nm band characteristic of the left- to right-handed helix transition. The intensity of the 222-nm CD band seems unaffected by the interaction with the side-band chromophore. The circular dichroism associated with the 330-nm band is much stronger in the right-handed conformation and is unaffected by increasing interactions between side chains. Interpretation in the light of the possible mechanism for optical activity of helical molecules seems to indicate that the 330-nm extrinsic effect is essentially due to the Condon, Altar, and Eyring (CAE) mechanism and μ-m perturbation (in Schellmann terminology) on the side-chain chromophore n, π* transition.     

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.     

Is DNA really a double helix?

Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to $\text{N}\text{10}$ (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling.     

β-Turn preference of tetrapeptide sequences as analyzed by CD spectra of their Dnp-pNA derivatives

The Dnp-pNA derivatives of more than 30 tetrapeptides were synthesized and subjected to CD measurements in MeOH. The CD spectra were analyzed on the basis of exciton chirality theory and were found to be useful for estimating β-turn preference of the parent tetrapeptides based on the following facts: (1) when a compound of the series takes the βturn conformation, a pair of strong CD bands with opposite sign are observed at 305 and 350 nm, and (2) when it is in a random or nonfolded conformation, no significant bands are observed at the wavelengths. The intensity of the 350-nm band reflects especially well the βturn preference. The CD spectra can be classified into two groups according to the sign of the band at 350 nm, and the groups seem to correspond to the type of βturn. By summarizing the results obtained, some generalizations concerning the sequence–conformation relationship are proposed.     

Quantum-mechanical theory of CD for infinite helical polymers

Quantum-mechanical equations are derived that are particularly well suited to actual computations of the CD for helical polymers. They make use of cyclic boundary conditions and helical symmetry, so that only two matrices with a size equal to the number of transitions considered need be diagonalized. The final equations are expressed directly in terms of monomer properties and helical parameters to invite the same input as earlier calculations, and are given as a rotational strength times a shape function for ease of comparison with the earlier work. The shape of the helix term is expressed as a derivative with respect to ω and depends on the distance between monomers along the helix axis. Other terms involving two electric transition dipoles depend on the distance from the helix axis to the transition center. These equations are directly comparable to the classical equations derived for cyclic boundary conditions and helical symmetry. We present an outline of the derivation and enough intermediate steps to clarify how the equations arise.     

Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers

We have designed experiments to characterise leech leukocytes that mediate inflammatory responses. Shortly after inflicting injury to the body wall in the presence of lipopolysaccharides, many cells resembling macrophages, NK cells and granulocytes of vertebrates and many invertebrates migrated to the lesioned area. Nuclei of migrating cells incorporated bromodeoxyuridine. Using human monoclonal antibodies, macrophage-like cells were positive for CD25, CD14, CD61, CD68, CD11b and CD11c. NK-like cells were positive for CD25, CD56, CD57 and CD16, and granulocytes were positive for CD11b and CD11c. In blots of leech extracts, the CD25 monoclonal antibody recognised a band of about 55 kD; the CD56 monoclonal antibody, two bands of about 140 and 210 kD; the CD57 monoclonal antibody, two bands of about 106 and 70 kD; the CD14 monoclonal antibody, a band of about 50 kD; the CD16 monoclonal antibody, a band of about 60 kD. CD61 and CD68 both recognised a band of about 110 kD; CD11b recognised a band of 200 kD, and CD11c, a band of 180 kD.     

Optical properties of polypeptides in the β-conformation

The rotational strengths and oscillator strengths of the nπ* band and ππ* exciton bands have been calculated for antiparallel and parallel β-structures of varying length and width. The results are compared with experiment and with previous theoretical treatments of β-structures. The generally good agreement of calculations on the antiparallel β-structure with experimental results on poly-L -lysine and poly-L -serine indicates that these systems are indeed in the antiparallel conformation. It is found that the exciton component strongest in absorption shifts to longer wavelengths as the width of an antiparallel structure increases, and it is suggested that the position of the ππ* absorption band may be a useful criterion of sheet width. The results also reconcile the linear dichroism measurements of Rosenheck and Sommer on poly-L -lysine films with an anti-parallel structure. Calculations on parallel β-structures indicate that the CD spectra of this form will be rather similar to that of the antiparallel form. However, the major absorption band in the antiparallel form is associated with a small positive CD band, while in the parallel form it coincides with a large negative CD band. Finally, it is pointed out that the large positive CD bands predicted for single-stranded parallel and antiparallel β-structures at about 200 mμ render unlikely the suggestion that random-coil polypeptides contain a substantial fraction of extended chain.