Alteration of 5S RNA conformation by ribosomal proteins L18 and L25.

The effects of ribosomal proteins L18, L25 and L5 on the conformation of 5S RNA have been studied by circular dichroism and temperature dependent ultraviolet absorbance. The circular dichroism spectrum of native 5S RNA is characterized in the near ultraviolet by a large positive band at 267 nm and a small negative band at 298 nm. The greatest perturbation in the spectrum was produced by protein L18 which induced a 20% increase in the 267 nm band and no change in the 298 nm band. By contrast, protein L25 caused a small decrease in both bands. No effect was observed with protein L5. Simultaneous binding of proteins L18 and L25 resulted in CD changes equivalent to the sum of their independent effects. The UV absorbance thermal denaturation profile of the 5S RNA L18 complex lacked the pre-melting behavior characteristic of 5S RNA. Protein L25 had no effect on the 5S RNA melting profile. We concluded that protein L18 increases the secondary, and possible the tertiary structure of 5S RNA, and exerts a minor stabilizing effect on its conformation while protein L25 causes a small decrease in 5S RNA secondary structure. The implications of these findings for ribosome assembly and function are discussed.     

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.     

Experimental test of absorption flattening correction for circular dichroism of particle suspensions

There are well established theoretical models for correction for absorption flattening of circular dichroism (CD) measurements on particle suspensions. However, these have not been directly tested experimentally. We describe a test system with the chiral tris(ethylenediamine)Co(III) complex dissolved in water trapped inside sephadex particles, suspended in 1-butanol. Independent measurements of particle size distribution, volume fraction, and the absorbance of the suspension are used to calculate the required CD correction. The corrected CD signal is found to agree rather well with that for the same amount of Co-complex dispersed uniformly throughout the sample cell. This holds for different particle volume fractions and Co-complex concentrations inside the particles. The correction seems to work despite a substantial scattering contribution to the absorbance, which is not considered in the theoretical models.     

Conformational changes in the active site of tryptophanase revealed by the circular dichroism method

Tryptophanase from E. coli displays positive CD in the coenzyme absorption bands at 337 and 420 nm. Breaking of the internal coenzyme-lysine imine bond upon reaction with hydroxylamine or amino-oxyacetate is accompanied by a strong diminution of the positive CD. Interaction of tryptophanase with L-threonine and beta-phenyl-DL-serine(threo form) leads to a decrease in absorbance at 337 nm and to an increase at 425 nm. This is associated with inversion of the CD sign, i.e. with disappearance of the positive CD in the 420-nm band and its replacement by a negative CD. L-Phenylalanine, alpha-methyl-DL-serine and D-alanine cause an increase in absorbance at 425-430 nm and a diminution of the positive CD in this band. In the presence of D-alanine and indole a negative CD appears in the 400-450 nm region. It is inferred that an external coenzyme-quasisubstrate aldimine is formed on interaction of the above amino acids with the enzyme. L-Alanine and oxindolyl-L-alanine evoke an intense narrow absorption band at 500 nm ascribed to a quinonoid intermediate; a positive CD is observed in this band. The dissymmetry factor delta A/A in the 500-nm band is much smaller than that in the absorption bands of the unliganded enzyme. Inversion of the CD sign on formation of the external aldimine and diminution of the dissymmetry factor in the quinonoid band indicate that reorientations of the coenzyme occur in the course of the catalytic action of tryptophanase.     

Interaction of tryptophanase with substrate analogs

Tryptophanase from Escherichia coli was studied with respect to its interactions with L-alanine, beta-chloro-L-alanine, L-phenylalanine, L-methionine, L-threonine, beta-phenyl-DL-serine (threo form) and also with a new tryptophan analog oxindolyl-L-alanine. Slow transamination of L-alanine in the active site of the enzyme was observed. Some evidence is presented which indicates that the side transamination reaction occurs during incubation of tryptophanase with an adequate substrate, beta-chloro-L-alanine. Absorption and circular dichroism (CD) spectra of the enzyme-quasisubstrate complexes have been recorded. Addition of beta-phenylserine and threonine to the enzyme induces a decrease of absorbance at 337 nm and an increase of absorbance at 420 nm. The spectral changes are associated with inversion of the CD sign, i.e. with disappearance of positive CD in the 420 nm band and appearance of negative CD in this band. It is inferred that beta-phenylserine and threonine form an external coenzyme-substrate aldimine which undergoes slow conversion to give a keto acid and the free enzyme. Addition of oxindolylalanine to tryptophanase results in the formation of an intense narrow absorption band at 504 nm with a shoulder at about 475 nm. This band belongs to a quinonoid intermediate. A positive CD is seen in the 504 nm band; the dissymmetry factor (delta A/A) in this band is much smaller than that in the absorption bands of the free enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism spectroscopy of fluorescent proteins

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.     

Optical studies on the structure of hemoglobin messenger ribonucleic acid

The secondary structure of hemoglobin mRNA (HbmRNA has been investigated by optical rotatory dispersion (ORD), circular dichroism (CD) and ultraviolet absorbance spectrophotometry. The dependence on temperature or reaction with formaldehyde of the CD and absorbance are characteristic of a structure with substantial base pairings at 20°C. The presence of Cotton effects in one of the major ultraviolet absorption bands indicates a highly ordered secondary structure. The UV hyperchromism on thermal denaturation is consistent with a value of 58% double helical content.     

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.     

Quantitative infrared spectroscopy of lipids in solution: III. Automated data collection and computer aided calculations of lipid analysis.

In the third part of this series, a data acquisition system is described which digitizes the analog signal from a commercial infrared spectrometer and records the data on paper tape. The paper tape is then processed off-line with a microcomputer and an infrared spectrum is printed. The correction routine for absorbance due to the O-H stretching band is discussed as well as the integration limits for calculation of integrated intensities of the C-H stretching band. The computer program is briefly described and statistical data on correlation between integrated intensity and lipid concentration given.     

Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles

Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 microm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.     

Proteolysis as a probe of thermal unfolding of cytochrome C

Recent hydrogen exchange experiments on native cytochrome c implicate a sequential unfolding pathway in contrast to a simple two-state process. We have studied the heat-induced unfolding of this protein by using spectroscopic measurements to detect changes in conformation and proteolytic enzyme digestion to identify regions of the protein that are labile. Several spectroscopic profiles were monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment of Trp 59, and absorbance at 420 nm, the Soret band of the heme. The apparent T_m values for these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent with population of an intermediate state in unfolding. We find a single strong region of cleavage at low temperature with retention of structure in each fragment. Proteins 30:435–441, 1998. © 1998 Wiley-Liss, Inc.     

Circular dichroism and proton magnetic resonance studies of random chain poly-L-lysine

The 218-nm peak, characteristic of the circular dichroism of randomly coiled poly-α-amino acids can be demonstrated in solutions of penta-L -lysine, α-glycyl-L -lysine, as well as poly-L -lysine. The thermal stability of the particular state that gives rise to this 218-nm band in the CD is similar for all three peptides. These results eliminate the possibility that poly-L -lysine forms a structure with long-range order in acidic aqueous solution since the stability of such a structure would be expected to be greater for a higher molecular weight polymer than for a pentamer. The intrinsic viscosity of poly-L -lysine of molecular weight 180,000 varies only slightly between 25 and 60°C. The proton magnetic resonance spectra of poly-L -lysine and penta-L -lysine are indistinguishable on the basis of the chemical shift of all resonances, their line widths, and the exchange rates of the N? H protons. This demonstrates that poly-L -lysine does not possess a cooperatively formed ordered structure in acidic solutions. A weak band at 238 nm is observed in the circular dichroism of poly-L -lysine and other peptides. It is suggested that the effects of change in temperature, salt concentration, or polymer on both the magnitude and position of the 238-nm band may be explained if it is assumed that it is a shoulder of a lower wavelength peak.     

Bovine lactoperoxidase and its recombinant: comparison of structure and some biochemical properties

Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.     

Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH.

The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions.     

A polycationic amine that induces unique conformational changes in poly(dA-dT) in low salt

Circular dichroism was used to examine alterations in the secondary structure of poly(dA-dT) X poly(dA-dT) upon binding polymer X, a polycationic CD probe for aspects of DNA structure. Stable complex formation is evidenced by increasing Tm and the appearance of large extrinsic bands in the greater than 300 nm, region which increase proportionally with r (ratio of polymer charge to DNa phosphate), in the range 0.0 to 0.32. At relatively low values of r (less than .32), CD spectra of the poly(dA-dT) X poly(dA-dT)-polymer X complex show a gradual non-cooperative inversion in the long wavelength portion (275 nm) of the intrinsic band in low salt solutions suggesting structural and conformational flexibility in poly(dA-dT) X poly(dA-dT) and further implicating polymer X as a potential probe for variations in DNA secondary structure. The dinucleotide repeat configuration of poly(dA-dT) X poly(dA-dT) is presumed to play a role in the observed intrinsic CD changes. NMR data support an "alternating B" conformation for the complex.     

Interpretation and Optimization of Absorbance and Fluorescence Signals from Voltage-sensitive Dyes

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density (OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.     

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).     

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

Spectral differences between rhodanese catalytic intermediates unrelated to enzyme conformation

Circular dichroism (CD) spectra and UV absorption spectra of two obligatory intermediates in rhodanese catalysis were compared. A broad CD band between 250 and 287 nm increased in a manner stoichiometrically related to the content of enzyme-bound persulfide. Titration of a sample of sulfur-substituted rhodanese (ES) with either cyanide or sulfite gave a stoichiometry that is consistent with one persulfide/molecule of rhodanese (Mr = 33,000). This result agrees with that determined by x-ray crystallography and a method based on quenching of intrinsic fluorescence. Cyanolysis of the persulfide in ES is accompanied by a decrease of UV absorption in the region between 250 and 300 nm. Cyanide titrations followed by the change in absorbance at 263, 272, and 292 nm gave the expected stoichiometry. The magnitude of the difference between the far UV-CD spectra of E and ES found here is smaller than reported previously. This variability suggests that the differences in the secondary structure of these intermediates may not be obligatorily related to the cyanolysis of the persulfide. This view is compatible with recent evidence which suggested that E and ES may be made different by structural relaxation events that occur outside of the catalytic cycle. Furthermore, the methods developed here will be useful in studies on the stability of the catalytic persulfide that has been suggested to be central in the mechanism of several enzymes important in sulfur metabolism.     

Studies on the interaction between antitrypanosome cis-DDP analogs with DNA

The nature of the conformational changes produced in DNA by cis-DDP analogs has been studied by physiochemical techniques. The UV spectra showed that the DNA undergoes bathochromic shifts accompanied by hyperchromic effects in reaction with specific analogs (cis-Pt(DDH) (mucobromic)2, cis-Pt(tranilcypromine)2Cl2 and cis-Pt(DDH) Cl2), while a different series of analogs (cis-Pt(DDH) (metafluorobenzoic)2 and cis-Pt(pentamidine)Cl2) induce a significative decrease in the absorbance at 258 nm. Moreover one of these analogs (cis-Pt(pentamidine)Cl2) causes strong stabilization of the double helix to heat denaturation. The CD spectra indicate moreover that cis-Pt(pentamidine)Cl2 modifies the secondary structure of the DNA in a significant way with an increase of the positive band and a decrease of the ellipticity of the negative band. The antitrypanosome activity of cis-Pt(pentamidine)Cl2 is probably due to inhibition of the intracellular parasites division in parasitized cells.