Tyrosine optical activity as a probe of the conformation and interactions of fibronectin

A very intense negative band is observed at ～ 183 nm in the CD spectrum of fibronectin from bovine plasma. This transition has not previously been reported, probably because it occurs in a spectral region that has not been readily accessible in earlier studies. At longer wavelength, the observed CD is very similar to spectra reported for human and chick material, having positive bands at ～230 and ～200 nm, and a negative band at ～215nm. The low molar ellipticity of the negative band ([θ] ≈ ?2.5 × 10³ deg cm² dmol^?1) suggests little α-helix or β-sheet structure. The new transition, and the two positive bands at higher wavelength, do not correspond to known transitions of the peptide backbone, but all three are present in the CD of N-acetyltyrosineamide. It is therefore suggested that the observed CD behavior of fibronectin arises predominantly from the optical activity of tyrosine side chains. The contribution of this side-chain optical activity to the CD of other proteins is discussed. On raising pH to ionize tyrosine residues, the positive CD band at ～230 nm is lost in both N-acetyltyrosineamide and in fibronectin. The spectral change is fully reversible in the model compound, but only partially reversible in fibronectin. From this evidence, and the magnitude of the 183-nm band, it is suggested that some or all of the tyrosine residues in fibronectin may be present within ordered domains. The possible role of S? S bonds in maintaining tertiary structure is discussed. The interaction of fibronectin with heparin is accompanied by a large increase in the 183-nm band and by slight enhancement of the negative band at 215 nm, consistent with some limited formation of β-sheet. Present results indicate that CD may be of considerable value in characterization of the molecular organization and biologically relevant interactions of fibronectins and of related glycoproteins of the extracellular matrix.     

The circular-dichroic properties of the ''Rieske'' iron-sulphur protein in the mitochondrial ubiquinol: cytochrome c reductase.

We have studied the c.d. spectra of the 'Rieske' iron-sulphur protein isolated from the ubiquinol: cytochrome c reductase (bc1 complex) of bovine heart mitochondria. Both the oxidized and the reduced form of the 'Rieske' protein display a series of well-resolved c.d. features resembling those reported for the 'Rieske'-type iron-sulphur protein purified from the bacterium Thermus thermophilus [Fee, Findling, Yoshida, Hille, Tarr, Hearshen, Dunham, Day, Kent & Münck (1984) J. Biol, Chem. 259, 124-133]. In particular, the difference spectra, reduced minus oxidized, of both proteins have a distinctive negative band at 497 nm. The c.d. features characteristic of the isolated 'Rieske' protein were found in the dichroic spectra of the whole bc1 complex in the region between 450 and 520 nm. The reduction of the enzyme by ascorbate or ubiquinol is accompanied by the formation of a negative band at about 500 nm that corresponds, in all its c.d. properties, to the specific dichroic absorption of the reduced 'Rieske' iron-sulphur protein.     

Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase.

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.     

Total optical activity of agarose: relation to observable transitions in the vacuum ultraviolet

The changes in optical activity that accompany and characterize the coil-helix and helix-coil transitions of agarose in aqueous solutions and gels have been investigated by combined quantitative analysis of data from vacuum ultraviolet circular dichroism (VUCD) and optical rotary dispersion (ORD). VUCD of agarose in the high-temperature coil state shows a single accessible Gaussian band centered at ～183 nm. In the helix state this band is blue-shifted by ～9 nm, and the intensity is increased by a factor of ～2.6. Spectra at intermediate temperatures can be fitted to within experimental error by linear combination of coil and helix spectra, the relative proportions required providing an index of the extent of conformational ordering. ORD spectra throughout the conformational transition have a common form and differ only in absolute magnitude. The temperature course of conformational ordering derived from ORD intensity is in close agreement with the values obtained from VUCD. In both the coil and helix states the accessible VUCD band is positive, while the overall ORD is negative, indicating strong negative CD activity at lower wavelength. The ORD contribution corresponding to the positive VUCD band was calculated by Kronig–Kramers transform, and it was subtracted from the total ORD to give the residual ORD from all other optically active transitions of the molecule. In both the coil and helix states, this residual ORD could be fitted to within experimental error by a single Gaussian CD band at ～149 nm. A negative band at this wavelength has been reported previously for agarose films, but the observed intensity, relative to that of the lower energy positive band, is substantially smaller than the fitted value under hydrated conditions. In both the coil and helix states the total optical activity of agarose, characterized by observed ORD spectra, can be matched to within experimental error by Kronig-Kramers transform of the 149-nm negative band and the smaller positive band at higher wavelength, with no necessary involvement of deeper-lying transitions. The significance of this conclusion for fundamental understanding of carbohydrate optical activity is discussed.     

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.     

Vacuum ultraviolet circular dichroism of galactomannans

The chiroptical behaviour of plant galactomannans has been investigated by a combined vacuum ultraviolet circular dichroism (v.u.c.d) and optical rotatory dispersion (o.r.d.) approach. The samples studied were from Cyamopsis tetragonolobus (guar), Caesalpin'ia spinosa (tara), and Ceratonia siliqua (carob), with galactose levels of 39, 25 and 19%, respectively. V.u.c.d. solid film spectra have been recorded down to 140 nm, and in all cases show a positive band at 169 nm, and a negative band at 149 nm whose relative intensity increases systematically with decreasing galactose content. In solution only the lower energy band is accessible, and has the same position and width as in the solid state but substantially greater amplitude. Residual o.r.d. behaviour, after subtraction of the contribution from the 169 nm band (calculated by Kronig-Kramers transform of fitted c.d. parameters) shows a single band of the same position and width as the high energy solid state transition. The amplitude of both transitions in solution varies linearly with galactose content, consistent with simple additivity of contributions from the two different residues. Extrapolation to 0 and 100% galactose yields molarr ellipticities (10³ x deg cm² dmol^?1) at 169 and 149 nm, respectively, of +14 and ?33 for mannan, and ?8 and ?80 for galactan. Reduction in the net intensity of both transitions in the solid state, and to a lesser degree in carob gels, is attributed to conformational restriction of galactose by chain packing, with consequent increase in c.d. intensity from these residues and hence greater cancellation of mannose backbone contributions.     

Vacuum ultraviolet circular dichroism of (1 → 6)-β-d-glucan

V.u.c.d. spectra recorded for freshly prepared aqueous solutions of (1 → 6)-β)-D-glucan(pustulan) contained a single positive band near 177 nm. This band was similar in position and magnitude to the single positive band observed in the spectrum of (1 → 6)-α-D-glucan (dextran). Pustulan solutions (20 mg/ml) were observed to gel with time at 10 C. Concurrently, a negative band at 190 nm developed in the pustulan v.u.c.d. spectrum followed by a blue shift of both bands with continued aging. Crystalline films of pustulan yield spectra which resembled the blue shifted spectra of aged gels. The time dependent development of the negative band was attributed to pustulan attaining a helical conformation in solution, and the blue shift to aggregation of helices, Na⁺ and Ca²⁺ were found to accelerate gelation presumably by decreasing the activity of the aqueous solvent.     

Circular dichroic spectra of elapid cardiotoxins

Cardiotoxins isolated from elapid snake venoms constitute a chemically homogeneous family of molecules. Within this group several biologically different subclasses exist. We report a comparative analysis of the structure of 20 cardiotoxins using circular dichroism, immunological methods and secondary-structure prediction. It is shown that cardiotoxins fall within two structural subclasses. Toxins of group I are characterized by (a) CD spectra having an intense positive band close to 192.5 nm and a negative trough at 225 nm with no positive band around 230 nm, (b) strong cross-reactivity with a polyclonal antiserum specific for Naja nigricollis toxin gamma and (c) a high tendency to form a reverse turn in the region of position 11. Toxins of group II are characterized by (a) CD spectra displaying a much weaker positive band at 192.5 nm, a negative band around 210 nm and a positive band at 230 nm, (b) little cross-reactivity with the aforementioned antiserum and (c) a high reverse-turn potential at position 31. It is suggested that the observed differences result from differing curvatures in the antiparallel beta sheet which constitutes the main secondary structure of cardiotoxins.     

Vacuum ultraviolet circular dichroism of poly(galacturonic acid), sodium polygalacturonate and calcium polygalacturonate

Vacuum ultraviolet circular dichroism spectra are reported for poly(galacturonic acid) solution and film, sodium polygalacturonate solution and film, and calcium polygalacturonate gel. In addition to the positive c.d. band near 208 nm previously observed, we find a pair of higher energy bands at 170 180 nm (negative) and 145 nm (positive). The low energy band, assigned to an $\text{n-π}{}^1$ carboxyl transition, is blue-shifted upon gelation or film formation.     

Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains

The structure of the lectin discoidin I has been studied by circular dichroism and fluorescence spectroscopy. A positive ellipticity band at 224 nm is detected in the CD spectrum of discoidin I. The fluorescence spectra show a defined shoulder at 325 nm that through acrylamide quenching has been associated with a displaced tryptophan residue partly buried in the discoidin I molecule. This tryptophan could also be responsible for the 224 nm positive band of the CD spectrum. These spectroscopic characteristics of discoidin I indicate the existence of structural homologies with fibronectin, where the optical activity of aromatic chromophores has been associated with the positive ellipticity band at 227 nm. The CD adjust parameters and theoretical secondary structure predictions show that discoidin I is a molecule with a low content of alpha-helix and beta-strand and high content of beta-turn structures, similar to other lectins.     

Spectroscopic studies on the structural organization of the lectin discoidin I: homologies with fibronectin

The structure of the lectin discoidin I has been studied by circular dichroism and fluorescence spectroscopy. A positive ellipticity band at 224 nm is detected in the CD spectrum of discoidin I. The fluorescence spectra show a defined shoulder at 325 nm that through acrylamide quenching has been associated with a displaced tryptophan residue partly buried in the discoidin I molecule. This tryptophan could also be responsible for the 224 nm positive band of the CD spectrum. These spectroscopic characteristics of discoidin I indicate the existence of structural homologies with fibronectin, where the optical activity of aromatic chromophores has been associated with the positive ellipticity band at 227 nm. The CD adjust parameters and theoretical secondary structure predictions show that discoidin I is a molecule with a low content of α-helix and β-strand and high content of β-turn structures, similar to other lectins.     

Shape,conformation and stability of fibronectin fragments determined by electron microscopy,circular dichroism and ultracentrifugation

Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K³ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.     

A study of the spectral and luminescence manifestations of the aggregation of thymine chromophores in polythymidylic acid aqueous solutions at room temperature in the context of photochemical information recording using thymine

The luminescence and excitation spectra of polythymidylic acid aqueous solutions at room temperature were studied. In addition to the previously described band at 338 nm, two new bands at 320 and 350 nm were recorded at various excitation wavelengths. An examination of the excitation spectra that had not been studied previously, as well as their comparison with the differential absorption spectra previously recorded during photodimerization, allowed us to interpret the band at 320 nm as the band of noninteracting chromophores; the band at 338 nm as the band of the most photochemically active, densely packed stacking dimmers (exciton splitting of absorption band of ～4000 cm^?1); and the band at 350 nm as the band of photochemically inactive large stacking aggregates (n ≥ 10, exciton splitting of ～8000 cm^?1). The changes in the optical density of the polythymidylic acid aqueous solution at γ = 270 nm after successive irradiation of the solution with light at 279 + 302 and 248 nm were studied. The reasons for their incomplete reversibility are discussed.     

The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

Initial characterization of the photosynthetic apparatus of "Candidatus Chlorothrix halophila," a filamentous, anoxygenic photoautotroph

“Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “Ca. Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ~6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “Ca. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.     

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.