Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

Novel circular dichroism spectroscopic approach for detection of ligand binding of proteins: Avidin as example

Various globular proteins having low or no α-helical content exhibit atypical far-ultraviolet (UV) circular dichroism (CD) spectra due to aromatic-aromatic residue and aromatic residue-peptide bond exciton coupling interactions. As a representative example of such proteins, far-UV CD spectra of chicken avidin were recorded before and after the addition of different small molecules. Intensity increase-decrease and/or wavelength shift of the positive CD peak of avidin at 228 nm were observed in the presence of various drugs, dyes, and natural compounds. The results were interpreted by exciton interactions between the aromatic residues of the biotin binding site and the substances bound to it. This novel, fast, microgram (μg) scale approach can be applied for detection of ligand binding of additional proteins displaying avidin-like far-UV CD spectra.     

A new, model-free calculation method to determine the coordination modes and distribution of copper(II) among the metal binding sites of multihistidine peptides using circular dichroism spectroscopy

A new calculation method to determine microscopic protonation processes from CD spectra measured at different pH and Cu(II):ligand ratios was developed and used to give the relative binding strengths for the three histidines of hsPrP(84-114), a 31-mer polypeptide modeling the N-terminal copper(II) binding region of human (homo sapiens) prion protein. Mutants of hsPrP(84-114) with two or one histidyl residues have also been synthesized and their copper(II) complexes studied by CD spectroscopy. The 1-His models were analyzed first, and the molar CD spectra for the different coordination modes on the different histidines were calculated using the general computational program PSEQUAD. These spectra were deconvoluted into the sum of Gaussian curves and used as a first parameter set to calculate the molar spectra for the different coordination modes (3N and 4N coordination) and coordination positions (His85, His96 and His111) of the 2-His peptides. The calculation method therefore does not require the direct use of CD spectra measured in the smaller peptide models. This is a significant improvement over earlier calculation methods. In the same runs, the stepwise deprotonation pK(mic) values were refined and the pH-dependent distribution of copper(II) between the two histidines was determined. The results revealed the high, but different copper(II) binding affinities of the three separate histidines in the following order: His85 < His96His111. The calculation also showed that molar CD spectra which belong to the same coordination mode and coordination position in different ligands have very similar transition energies but different intensities. For this reason, direct transfer of molar CD spectra between different ligands may be a source of error, but the pK(mic) values and the copper(II) binding preferences are transferable from the 2-His peptides to the 3-His hsPrP(84-114).     

Circular dichroism studies of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with DNA

The circular dichroism spectra of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with calf thymus DNA have been measured over a range of drug/DNA ratios. The similarity of the CD spectra of bound chromomycin and mithramycin suggests that they have very similar binding sites, which produce strong effects on the CD spectra of the bound drugs, and remove the differences arising from local stereochemistry in the free drugs. It was found that it was not possible to predict whether the antibiotics intercalated, from studies of the CD spectra alone, even when comparisons were made with the CD spectra of aminoacridine–DNA complexes with intercalating or nonintercalating ligands.     

Empirical rules for rationalising visible circular dichroism of Cu2+ and Ni2+ histidine complexes: applications to the prion protein

A natively unfolded region of the prion protein, PrP(90-126) binds Cu(2+) ions and is vital for prion propagation. Pentapeptides, acyl-GGGTH(92-96) and acyl-TNMKH(107-111), represent the minimum motif for this Cu(2+) binding region. EPR and (1)H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His(96) and His(111) to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu(2+) visible CD spectra involving histidine and amide main-chain coordination.     

Interaction of rhein with human serum albumin investigation by optical spectroscopic technique and modeling studies

The binding of rhein with human serum albumin (HSA) has been studied in detail by spectroscopic method including circular dichroism (CD), Fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters for the reaction have been calculated according to Scatchard equation at different temperatures. The plots indicated that the binding of HSA to rhein at 303, 310 and 318 K is characterized by one binding site with the affinity constant K at (4.93+/-0.16)x10(5), (4.02+/-0.16)x10(5) and (2.69+/-0.16)x10(5) M-1, respectively. The secondary structure compositions of free HSA and its rhein complexes were estimated by the FT-IR spectra. FT-IR and curve-fitted results of amide I band are in good agreement with the analyses of CD spectra. Molecular Modeling method was used to calculate the interaction modes between the drug and HSA.     

Classification of CD and absorption spectra in the Soret band of H(2)TMPyP bound to various synthetic polynucleotides

The binding mode of porphyrins, namely meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (H(2)TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H(2)TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.     

Effects of ssDNA sequences on non-sequence-specific protein binding

The circular dichroism (CD) spectra of single-stranded DNAs (ssDNAs) are significantly perturbed by the binding of single-stranded DNA binding proteins such as the Ff bacteriophage gene 5 protein (g5p) and the A domain of the 70 kDa subunit of human replication protein A (RPA70-A). These two proteins have similar OB-fold secondary structures, although their CD spectra at wavelengths below 250 nm differ greatly. The spectrum of g5p is dominated by a tyrosyl L(a) band at 229 nm, while that of RPA70-A is dominated by its beta secondary structure. Despite differences in their inherent spectral properties, these two proteins similarly perturb the spectra of bound nucleic acid oligomers. CD spectra of free, non-protein-bound ssDNAs are dependent on interactions of the nearest-neighboring nucleotides in the sequence. The CD spectra (per mol of nucleotide) of simple repetitive sequences 48 nucleotides in length and containing simple combinations of A and C are related by nearest-neighbor equations. For example, 3 x Deltaepsilon[d(AAC)(16)] = 3 x Deltaepsilon[d(ACC)(16)] + Deltaepsilon[d(A)(48)] - Deltaepsilon[d(C)(48)]. Moreover, nearest-neighbor equations relate the spectra of ssDNAs when they are bound by g5p, indicating that each type of perturbed nearest neighbor has a similar average structure within the binding site of the protein.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A.

Circular dichroism (CD) spectra of class II MHC peptides revealed the alpha-helical conformation of superantigen-binding peptides I-A beta b(60-90), I-A beta b(65-85), and I-A alpha b(51-80), but not the nonbinding peptide I-A beta b(80-100). These CD spectra provide biophysical evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A (SEA). Alanine-substituted analogs of the SEA binding-site peptide, I-A beta b(65-85), were used to implicate beta-chain residues 72 and 80 in class II MHC-SEA binding. The data support SEA binding away from the class II antigen binding cleft along the faces of the alpha-helices.     

Binding of wogonin to human gammaglobulin

The binding of wogonin to HGG was studied by spectroscopic method including circular dichroism (CD), fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters and the thermodynamic parameters for the reaction have been calculated according to Sips method and Gibbs-Helmholtz equation, respectively, at different temperatures. AutoDock3.05 program was used to calculate the interaction modes between the drug and HGG. The Sips plots indicated that the binding of HGG to wogonin at 297, 304, 310 and 317 K is characterized by two binding sites with the average affinity constant Ko at 2.102x10(4), 2.078x10(4), 1.956x10(4) and 1.931x10(4), respectively. The binding process was exothermic, spontaneous and entropy driven, as indicated by the thermodynamic analyses, and the major part of binding energy is electrostatic interaction accompanied by hydrophobic interaction and hydrogen bond. The secondary structure compositions of free HGG and its wogonin complexes were estimated by the FT-IR spectra and the curve-fitted results of amide I band, which are in good agreement with the analyses of CD spectra. Furthermore, the average binding distance between wogonin and HGG (5.60 nm) was obtained on the basis of the theory of F?rster energy transfer.     

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.     

Species-dependent stereoselective drug binding to albumin: a circular dichroism study

Drug binding to albumins from different mammalian species was investigated to disclose evidence of species-dependent stereoselectivity in drug-binding processes and affinities. This aspect is important for evaluating the reliability of extrapolating distribution data among species. The circular dichroism (CD) signal induced by drug binding to the albumins [human serum albumin (HSA), bovine serum albumin (BSA), rat serum albumin (RSA), and dog serum albumin (DSA)] were measured and analyzed. The binding of selected drugs and metabolites to HSA significantly differed from the binding to the other albumins in terms of affinity and conformation of the bound ligands. In particular, phenylbutazone, a marker of site one on HSA, showed a higher affinity for binding to BSA with respect to RSA, HSA, and DSA, respectively. In the case of diazepam, a marker of site two on HSA, the affinity decreased in order from HSA to DSA, RSA, and BSA. The induced CD spectra were similar in terms of energy and band signs, suggesting almost the same conformation for the bound drug to the different albumins. Stereoselectivity was high for the binding of ketoprofen to HSA and RSA. A different sign was observed for the CD spectra induced by the drug to the two albumins because of the prevalence of a different conformation of the bound drug. Interestingly, the same induced CD spectra were obtained using either the racemic form or the (S)-enantiomer. Finally, significant differences were observed in the affinity of bilirubin, being highest for BSA, then decreasing for RSA, HSA, and DSA. A more complex conformational equilibrium was observed for bound bilirubin.     

Spectroscopic investigation of the interaction of the toxicant, 2-naphthylamine, with bovine serum albumin

The mechanism of interaction between bovine serum albumin (BSA) and 2-naphthylamine (2-NA) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra, and UV-vis spectroscopy. It was proved from fluorescence spectra that the fluorescence quenching of BSA by 2-NA was a result of the formation of complex between 2-NA and BSA, and the binding constants (K(a) ) as well as the numbers of binding sites for 2-NA in BSA were determined according to the modified Stern-Volmer equation. The results of synchronous fluorescence and CD spectra demonstrated 2-NA could decrease the amount of α-helix of BSA, leading to the loosening of protein skeleton. UV-vis spectroscopy and resonance light scattering spectra (RLS) results also suggested the conformation of BSA were changed and the BSA aggregation occured, which could induce toxic effects on the organism.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding

Circular dichroism, CD, spectra can be used to gain information about quadruplex structures of DNAs as well as the effects of sequence, cations, chemical modification and ligand binding on quadruplex structure. There is not yet a validated approach to calculate a CD spectrum from a quadruplex structure nor is their one to go from a CD spectrum to a structure. However, it is possible to empirically correlate CD spectra features with quadruplex structural type in many cases. In this article four case studies are presented to indicate the strengths and limitations of CD in investigations of the properties of quadruplex structures formed by telomere repeat sequences. The case studies include determination of the quadruplex structural type present as a function of potassium concentration, the effect of sequence on the equilibrium between quadruplex structural types as a function of potassium concentration, the effect of ligand binding on quadruplex structure and the effect of 5' phosphorylation on quadruplex structural type.     

藏红花凝集素分子化学修饰与其活性的关系

对甘露糖专一性结合藏红花凝集素 (Crocussativuslectin ,CSL)分子进行化学修饰 ,测定酿酒酵母 (S .cerevisiae)凝集活性和寡糖专一性结合活性的变化 .实验结果表明 ,Cys的修饰与活性无关 ,Arg、Tyr和His的修饰降低了CSL分子的酵母凝集活性和寡糖结合活性 ,但对CSL的CD光谱无显著影响 ,表明其为凝集素的活性氨基酸残基 .Glu和Asp的化学修饰可使CSL的凝集活性大幅度降低 ,与特异性寡糖的亲和力增大 ,CD光谱变化明显 ,提示CSL分子中的Glu和Asp对其空间结构影响较大 ,氨基酸羧基的修饰导致CSL构象改变 ,蛋白与寡糖的结合位点暴露 ,可有效结合的位点数增加     

Circular dichroism spectra and ethidium bromide binding of 5-deoxybromouridine-substituted chromatin.

Changes in CD spectra of 5-deoxybromouridine (brUdRib)-substituted chromatin depend on the extent of thymidine replacement by brUdRib. With 20 and 14 per cent replacement, a blue shift and a marked increase in positive ellipticity are reported in the CD spectra of brUdRib-substituted chromatin, while with 4.9 per cent replacement little effect is noticed. BrUdRib-substitution does not affect the number of ethidium bromide primary binding sites of chromatin.     

Cooperative binding of hexadecyltrimethylammonium chloride and sodium dodecyl sulfate to cytochrome c and the resultant change in protein conformation

The binding of hexadecyltrimethylammonium chloride (HTAC) and sodium dodecyl sulfate (SDS) to cytochrome c was determined by potentiometric titration and the corresponding changes in protein conformation by circular dichroism (CD). The binding isotherms were biphasic; about 20 surfactant cations or anions were bound to cytochrome c in the first phase. Another 30 or so HTA+ ions were bound in the second phase, which was below the critical micelle concentration of the surfactant, but the binding of dodecyl sulfate ions in the second phase increased sharply near the critical micelle concentration. The binding of both surfactants was highly cooperative and was endothermic; the data in the first phase fitted the Hill plot. The corresponding change in the secondary structure of cytochrome c was small; the CD spectra in the ultraviolet region showed a moderate increase in the helicity in HTAC solution and some changes in conformation in SDS solution. However, the CD spectra for the Soret band indicated a marked change in the local conformation around the heme.