Photophysics of ANS. V. Decay modes of ANS in proteins: the IFABP-ANS complex

The fluorescence properties of ANS as bound to proteins are treated. Several points of view concerning the origin of these properties are reviewed and synthesized into one framework. On proteins where the quantum yield (QY) is appreciable, as in organic solvents, the preferred conformation of ANS is often with the phenyl ring nearly (65 degrees -85 degrees ) orthogonal to the naphthalene. The major consequence of this geometry is water exclusion from the critical zone of ANS at which the largest amount of solvent dipolar relaxation originates. This, in turn, leads to a depression of the rate of electron transfer to the surroundings, together with other effects, as noted in the literature and in our lab. Alternative quenching pathways for ANS on the protein vs in water are also elucidated.     

Photophysics of ANS. IV. Electron transfer quenching of ANS in alcoholic solvents and mixtures

ANS is observed to decay from the fluorescent state with distributed kinetics in nearly pure ethanol solvent, notwithstanding that in mixed ethanol/water solvents the decay is discrete and biexponential. The origin of this behavior is investigated. In particular, a theory of electron transfer theory in the adiabatic regime is adduced, with specific involvement of solvent cage structure in the form of the solvent-electron polaron wave function. Properties of various polarons for various solvent systems are predicted and, for the case of ethanol and cyclohexanol, employed to generate the observed Arrhenius-type decay parameters in a quantitative fashion.     

Photophysics of ANS. II: Charge transfer character of near-UV absorption and consequences for ANS spectroscopy

We continue our investigation of the photophysics of 1,8-anilinonaphthalenesulfonate in protein and solvent systems. In this report, we concentrate on the nature of the excited states as observed in UV spectra. We develop a fairly general formalism for handling the coupled transitions we observe in the partial systems aniline and naphthalene. We assign one of the near-UV transitions, which is more clearly discernible in congeners of 1,8-ANS, but still present in 1,8-ANS as we postulate it, to a charge-transfer band. The other transition is from aniline itself. The expected energies of these now coupled bands in anilinonaphthalene and ANS are calculated, and the transition dipole moment for these transitions is derived.     

Dehydro-dermorphins. III. Circular dichroism investigation of conformation in solution

Dehydropeptide analogs of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and N-terminal fragments containing one or two dehydrophenylalanine residues in the 3rd and/or 5th position, have been investigated by means of CD spectroscopy. The results indicate that the above dehydropeptides can adopt different conformations in alcohol and water solutions. In methanol and trifluoroethanol, the CD spectra are mainly consistent with the presence of folded structures, probably stabilized by intramolecular hydrogen bonds. In water, conversely, CD data indicate disruption of ordered structures and formation of preferentially extended flexible conformations. Models of the involved folded structures are tentatively proposed, taking into account the geometric features of dehydro residues and their tendency to favor hydrogen-bonded 10-membered rings.     

Photophysics of ANS. I. Protein-ANS complexes: Intestinal fatty acid binding protein and single-trp mutants

We continue investigations into the physical chemistry of intestinal fatty acid binding protein, I-FABP, and its interaction with ANS and other ligands [cf references [Kirk, W., E. Kurian, and F. Prendergast. 1996. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys. J. 70: 69-83., Kurian, E., W. Kirk, and F. Prendergast. 1996. Affinity of fatty acid for rRat intestinal fatty acid binding protein: Further examination. Biochemistry. 35:3865-74]. The photophysics of the wt protein is compared with that in two mutants which lack respectively one or the other of two trp moieties, one of which, trp 82, is located near the binding region for the polar head group of ligands. These studies afford a look into how the fluorescence of the wt protein is established, that is, as an almost direct sum of the fluorescence of the two individual trp residues, and how this fluorescence is quenched upon binding to ANS. Though we have access to all the relevant spectroscopic and geometric information necessary to specify in detail the Foerster-Dexter energy transfer model, the quenching process is not explicable in terms of very-weak coupling, as is usually assumed in fluorescence studies in protein systems, but in terms of a stronger effect which goes beyond the simple very-weak dipole:dipole formalism. The quenching of trp emission by bound ANS is not as great as that anticipated by ordinary resonance energy transfer, neither is the quenching observed in the reduced lifetimes of the trp emission upon ANS binding as great as that observed in steady-state intensity. However the observed steady-state quenching is explicable in terms derived from the lifetime measurements, together with observed spectral band shifts, by the exciton coupling model we invoke here.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Circular dichroism of some mononucleosides.

The circular dichroism (CD) and absorption spectra of uridine, thymidine, purine ribonucleoside, and the four adenine derivatives 2′-deoxyadenosine, adenosine, adenosine-3′,5′-cyclic phosphate, and arabinosyl adenine were measured in water at pH 7 and pH 2. The absorption and CD spectra of the pyrimidines were simultaneously fitted to four Gaussian bands, and the dipole and rotational strengths of the electronic transitions determined. Adenine-derivative CD spectra were determined by computer averaging six runs. The spectra showed CD bands at 268, 226, 209, and 195 nm. The band at 226 nm probably is an n–π* transition; the band at 209 nm cannot be detected without a computer. The CD and absorption spectra of purine ribonucleoside indicate three transitions in the 230–310-nm region.     

Circular dichroism of soybean leghemoglobin.

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.     

Circular dichroism and DNA secondary structure.

The change in average rotation of the DNA helix has been determined for the transfer from 0.05 M NaCl to 3.0 M CsCl, 6.2 M LiCl and 5.4 M NH4Cl. This work, combined with data at lower salt from other laboratories, allows us to relate the intensity of the CD of DNA at 275 nm directly to the change in the number of base pairs per turn. The change in secondary structure for the transfer of DNA from 0.05 M NaCl (where it is presumably in the B-form) to high salt (where the characteristic CD has been interpreted as corresponding to C-form geometry) is found to be -0.22 (+/- 0.02) base pairs per turn. In the case of mononucleosomes, where the CD indicates the "C-form", the change in secondary structure (including temperature effects) would add -0.31 (+/- 0.03) turns about the histone core to the -1.25 turns estimated from work on SV40 chromatin. Accurate winding angles and molar extinction coefficients were determined for ethidium.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Circular dichroism studies of ampullosporin-A analogues.

Ampullosporin A (AmpA), a 15mer peptalbol containing seven Aib residues is able to induce pigmentation on Phoma destructiva and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformational flexibility and biological activities. The analogues were obtained by modifying the N- and C-termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues were replaced by Ala and/or Ac6c. To estimate the helicity of the analogues, the CD spectrum of AmpA recorded in acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes in organic solvents with the ratio between two negative band intensities R = [theta]n-pi*/[theta]pi-pi* < 1. In acetonitrile, most of the analogues adopted a 70%-85% helical structure, which was higher than the average of 40%-60% obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a R ratio > 1 and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

Circular dichroism study of polyriboxanthylic acid.

We report in the present paper the circular dichroism spectra of poly(X) at different pH and temperature values. The spectra are characteristic of three stable forms of poly(x) in the pH range of protonation of xanthosine. An electrostatic barrier is proposed to account for the hysteresis and metastability observed in a certain pH range. Some results on oligo(X) at basic pH are also presented. Poly(X) at basic pH is investigated also by hydrodynamic techniques.     

Circular dichroism of DNA frayed wires.

Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A15G15) in aqueous solutions containing 5 mM MgCl2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A15) from that of d(A15G15), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10 degrees C and 60 degrees C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T15) to the solution, complexes with the adenine portion of the d(A15G15) frayed wires are formed. Subtraction of d(A15):d(T15) spectra measured at several temperatures from those of the d(A15G15):d(T15) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A15):d(T15) duplex is identical to that of the unbinding of d(T15) from d(A15G15):d(T15) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide.     

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region. In spite of the close geometric similarity of the beta turns of these two groups of peptides, their CD spectra are quite distinct. Furthermore, comparison of our data with the CD spectra of published models for beta-turn structures suggests that it may not be possible to characterize the contribution of all beta turns to the CD spectra of proteins by a single model curve. the CD spectra of model beta turns will be more useful in characterizing the folding of oligopeptides and sequence polypeptides, where a single type of turn is present.     

Circular dichroism of chromium(III) hexadentate edta-type complexes. Part II. Ethylenediaminetetra-3-propionatochromate(III) ion

The hexadentate Cr(III) complex with ethylenediaminetetra-3-propionate (edtp) ion has been prepared and resolved. Infrared, electronic absorption and CD spectra were used to characterize the [Cr(edtp)]⁻ complex. The CD data in the region of the d-d transitions are discussed in comparison with those of other edta-type Cr(III) complexes of known configuration. The (+)₅₈₉₋[Cr(edtp)]⁻ complex, having a positive (dominant) CD peak in the first spin-allowed d-d absorption band region, is tentatively assigned the Δ configuration.