Showing papers on "Circular dichroism published in 1995"
TL;DR: Circular dichroism is a spectroscopic method which depends on the fact that certain molecules interact differently with right and left circularly polarized light and can provide information about the secondary structure of proteins and nucleic acids and about the binding of ligands to these types of macromolecule.
Abstract: Publisher Summary Circular dichroism (CD) is a spectroscopic method which depends on the fact that certain molecules interact differently with right and left circularly polarized light. Circularly polarized light is chiral—that is, it occurs in two nonsuperimposable forms that are mirror images of one another. To discriminate between the two chiral forms of light, a molecule must be chiral, including the vast majority of biological molecules. A method that can discern the subtle differences between non superimposable mirror image molecules (enantiomers) must be highly sensitive to the three-dimensional features of molecules—that is, to conformation. Binding of ligands or protein-protein and protein–DNA interactions can also alter the circular dichroism spectrum of the protein and/or nucleic acid. These changes in CD can be used to determine equilibrium constants, and they can also provide evidence for conformational changes. Thus, CD can provide information about the secondary structure of proteins and nucleic acids and about the binding of ligands to these types of macromolecule.
941 citations
TL;DR: It is shown that β-lactoglobulin, which consists predominantly of β-sheets, exhibited a markedly high propensity to form an α–-helical structure in the presence of TFE, as measured by far-UV circular dichroism.
429 citations
TL;DR: The algorithm correctly calculates the average helical behaviour in solution of 423 peptides analysed by circular dichroism and it describes the helicity at a residue level, as found when comparing the prediction for each amino acid residue with the data derived from nuclear magnetic resonance studies.
357 citations
TL;DR: A model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides is proposed, which could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross-roads in cellular signalling.
Abstract: Phosphatidylinositol bisphosphate has been found to bind specifically to pleckstrin homology (PH) domains that are commonly present in signalling proteins but also found in cytoskeleton. We have studied the complexes of the beta-spectrin PH domain and soluble inositol phosphates using both circular dichroism and nuclear magnetic resonance spectroscopy, and X-ray crystallography. The specific binding site is located in the centre of a positively charged surface patch of the domain. The presence of 4,5-bisphosphate group on the inositol ring is critical for binding. In the crystal structure that has been determined at 2.0 A resolution, inositol-1,4,5-trisphosphate is bound with salt bridges and hydrogen bonds through these phosphate groups whereas the 1-phosphate group is mostly solvent-exposed and the inositol ring has virtually no interactions with the protein. We propose a model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides. They could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross-roads in cellular signalling.
326 citations
TL;DR: Peptide penetration into the lipid membrane and peptide aggregation at the membrane surface are proposed as possible mechanisms to explain the lipid-induced random coil<-->beta-structure transition.
318 citations
TL;DR: Using CD spectroscopy, the effect of Cu2+ on the secondary structure of synthetic peptides Octa4 and Hexa4 corresponding to tetra-repeats of the octapeptide of mammalian PrP and the hexapeptides of chicken PrP is investigated.
315 citations
230 citations
TL;DR: It is argued that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer.
Abstract: Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.
226 citations
TL;DR: CD spectra provide an important means of characterizing differences in the conformations of DNA, RNA, and hybrid duplexes or triplexes having analogous sequences.
Abstract: Absorption and CD measurements of complementary oligomers and mixtures are described. The concentrations of oligomers may be estimated from absorption measurements and nearest-neighbor calculations of molar extinction coefficients. Interactions between complementary strands in mixtures can lead to obvious differences between measured CD spectra and the average of the spectra of the individual strands. CD spectra also allow an assessment of whether the individual strands are in self-complexes, which could compete with duplex or triplex formation. Isodichroic and isoabsorptive points provide important indicators of the stoichiometry of the strands in base-paired complexes. CD spectra provide an important means of characterizing differences in the conformations of DNA, RNA, and hybrid duplexes or triplexes having analogous sequences.
224 citations
TL;DR: The structures of synthetic peptides corresponding to regions of putative secondary structure in the cellular prion protein PrPC were studied as models for the conformational transition that features in the formation of the pathogenic isoform, PrPSc.
Abstract: The structures of synthetic peptides corresponding to regions of putative secondary structure in the cellular prion protein PrPC were studied as models for the conformational transition that features in the formation of the pathogenic isoform, PrPSc. Transgenetic studies argue that these PrP isoforms interact during the formation of PrPSc, which involves the unfolding of one or more helices of PrPC followed by refolding into beta-sheets. PrP residues 109-122 (H1), which were predicted to be alpha-helical, form beta-sheets in aqueous buffers, while the longer peptide 104-122 (104H1) and also peptide 129-141 (H2) have coil or alpha-helical structures in solution. Both 104H1 and H2 were converted into beta-sheets upon interaction with H1, as monitored by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy. The conversion was sequence-specific since mouse (Mo) H1, which differs from Syrian hamster (SHa) at two residues, was inefficient at converting SHa104H1 into the beta-sheet form. In buffers containing 10% acetonitrile, 104H1 was converted into the beta-sheet form by addition of as little as 1% H1. In addition, A beta 11-25 and A beta 25-35 peptides with similar physical properties to H1 were incapable of converting H2 into the beta-sheet form. How well these studies approximate the structural transitions in PrP that underlie the replication of prions remains to be established.
216 citations
TL;DR: NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding, as seen in the refolding of apo bovine α-lactalbumin.
Abstract: The refolding of apo bovine alpha-lactalbumin has been monitored in real time by NMR spectroscopy following rapid in situ dilution of a chemically denatured state. By examining individual resonances in the time-resolved NMR spectra, the native state has been shown to emerge in a cooperative manner from an intermediate formed in the dead-time of the experiments. The kinetics of folding to the native state are closely similar to those observed by stopped-flow fluorescence and near-UV circular dichroism. The NMR spectrum of the transient intermediate resembles closely that of the well characterized stable molten globule state formed at low pH. The results suggest that NMR can play a key role in describing at an atomic level the structural transitions occurring during protein folding.
TL;DR: The results unequivocally demonstrate that prothymosin is a monomer under physiological conditions, which raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides.
Abstract: Prothymosin is an acidic protein with an unusual amino acid composition. Though its exact function is not yet known, its high evolutionary conservation and wide tissue distribution suggest an essential biological role. Its physical state, which is controversially discussed in previous publications, was investigated using small-angle X-ray scattering, dynamic light scattering, mass spectrometry, and circular dichroism (CD). Our results unequivocally demonstrate that prothymosin is a monomer under physiological conditions. The protein adopts a random coillike conformation but exhibits persistence of direction and curvature. No regular secondary structure is detectable by CD. The Stokes radius, Rs = 3.07 nm, and the radius of gyration, RG = 4.76 nm, are 1.77 and 3.42 times larger, respectively, than those expected for a compactly folded protein consisting of 109 amino acid residues. A remarkable amount of secondary structure is formed only in the presence of trifluoroethanol at low pH. The finding that a biologically active protein molecule with 109 amino acid residues adopts a random coil conformation under physiological conditions raises the question whether this is a rare or a hitherto-overlooked but widespread phenomenon in the field of macromolecular polypeptides.
TL;DR: The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration, showing single-exponential behavior, consistent with a unimolecular reaction.
Abstract: Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.
TL;DR: It is suggested that perturbation of the packing environment of the highly conserved residues is a possible mechanism for triggering the conversion of PrPcto PrPsc where α-helices appear to be converted into β-sheets.
TL;DR: Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character, suggesting that alpha-helices are important for transactivation in vivo.
Abstract: A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.
TL;DR: This paper showed that the intrinsic helical propensity of a peptide fragment elucidated by the addition of 2,2,2-trifluoroethanol (TFE) is not necessarily related to the secondary structure in the native state.
TL;DR: It is shown that A beta 1-40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties and the data suggest that the determinant factor of A beta fibrillsogenesis is the secondary structure adopted by the peptide in its soluble state.
TL;DR: It is concluded that the conformations of NK‐lysin and SP‐B are similar, and strictly conserved structural features with implications for helix topology and lipid interactions are observed.
TL;DR: The change in the secondary structure of T4 lysozyme upon adsorption to silica particles was studied with circular dichroism and the rate of conformational change differed among the three proteins and was fastest for the tryptophan mutant.
TL;DR: It is shown that the beta-hairpin structure is further stabilized in 30% trifluoroethanol and destabilised in the presence of 6 M urea, though some folded structure persists even in these highly denaturing conditions.
Abstract: The solution structure of the isolated fragments 1–20 (β-hairpin), 21–40 (α-helix) and 41–56 (β-hairpin), corresponding to all the secondary structure elements of the protein G B1 domain, have been studied by circular dichroism and nuclear magnetic resonance techniques. In the protein G B1-(1–20) fragment turn-like folded structures were detected in water though low populated. In the presence of 30% aqueous trifluoroethanol there is a complex conformational behaviour in which a helical structure at the N-terminal half is formed in equilibrium with random and native-like β-hairpin structures. The peptide corresponding to the α-helix is predominantly unstructured in water, while in 30% trifluoroethanol it highly populates a native α-helical conformation, including a (i,i+5) interaction between hydrophobic residues at its C-terminus. The third peptide was previously reported to form a monomeric native β-hairpin structure in water [Blanco, F. J., Rivas, G. & Serrano, L. (1994a) Nature Struc. Biol. 1, 584–590]. We show in this work that the β-hairpin structure is further stabilized in 30% trifluoroethanol and destabilised in the presence of 6 M urea, though some folded structure persists even in these highly denaturing conditions. The conformational properties of these peptides suggests that the second β-hairpin could be an important folding initiation site on which the rest of the chain folds. Reconstitution experiments failed to show evidence of interaction between the peptides. Algorithms designed to predict the helical and extended conformations of peptides in aqueous solution successfully describe the complicated behaviour of these peptides. Comparison of the predicted and the experimental results with those for a structurally related protein, ubiquitin, shows very strong similarities, the main difference being the switch of the most stable β-hairpin from the N-terminus in ubiquitin to the C-terminus in protein G.
TL;DR: The refolding from denaturing conditions of a small four-helix bundle, the acyl-coenzyme A binding protein, has been investigated by utilizing an array of fast-reaction techniques, indicating that the different elements of structure form effectively concomitantly.
Abstract: The refolding from denaturing conditions of a small four-helix bundle, the acyl-coenzyme A binding protein, has been investigated by utilizing an array of fast-reaction techniques. Stopped-flow tryptophan fluorescence for measuring the incorporation of aromatic residues into the protein core and far- and near-ultraviolet circular dichroism to measure the formation of secondary and tertiary structure, respectively, together with the formation of persistent structure measured by hydrogen exchange pulse labeling experiments analyzed by electrospray ionisation mass spectrometry all show that 90% of the acyl-coenzyme A binding protein molecules achieve their fully folded and active, native state with a time constant of less than 5 ms at 25 degrees C and of ca. 30 ms at 5 degrees C. The kinetic parameters measured by the different techniques are closely similar, indicating that the different elements of structure form effectively concomitantly. There is no evidence for a significant population of any partially structured intermediate states, and the kinetics are identical whether refolding occurs from an unfolded state generated either by low pH or by addition of guanidine hydrochloride. The kinetics of both refolding and unfolding are monophasic processes for practically 90% of the molecules, and can be described by a two-state model. The results add to our knowledge of the folding scheme of different structural motifs and are discussed in terms of current views of the mechanisms of protein folding.
TL;DR: Novkova et al. as mentioned in this paper introduced porphyrins as long-range interacting chromophores that extend the applicability of the exciton-coupled circular dichroic method to configurational studies of molecules with remote stereogenic centers and possibly to conformational studies for biopolymers.
Abstract: We introduce porphyrins as long-range interacting chromophores that extend the applicability of the exciton-coupled circular dichroic method to configurational studies of molecules with remote stereogenic centers and possibly to conformational studies of biopolymers. The interaction between excited states of chromophores in chiral environments give circular dichroism (CD) curves with split Cotton effects, Le., exciton-coupled CD.' In this method for determining the absolute stereochemistry of organic molecules in solution, the signs and shapes of the characteristic CD curves are defined by the absolute skewness of interacting chromophores.' The extent of chromophoric coupling, Le., the amplitudes of split Cotton effects, is inversely proportional to the square of interchromophoric distance2 and proportional to the square of extinction coeff i c i en t~~ of the coupled chromophores. Therefore, the intensity of chromophoric absorptions is of prime importance in increasing the sensitivity over the large distance between interacting transition moments. Furthermore, when the original substrate contains a chromophore, new chromophores with bathochromic intense absorbances, E = 31 000-58 000, may be used to deliberately avoid coupling with preexisting chromophore^.^ For the purpose of developing intense red-shifted chromophores, we had investigated cyanine dye chromophores; although unique from a spectroscopic viewpoint$b they were unsuited for practical applications due to their instability, nontrivial microscale preparation, etc. This led to the introduction of other redshifted chromophores which have wide and diverse applicab i l i t i e ~ . ~ ~ . ~ In the following, we show that porphyrins further enhance the sensitivity of the exciton-coupled CD method by almost 10-fold and extend the applicability of the method to molecules that could not be studied so far. Thus, 5-substituted 10,15,20-triphenylporphyrins, e.g., 1, with their intense redshifted Soret band at 414 nm, E = 350 000: provide powerful exciton-coupled CD chromophores for absolute configurational studies of natural products with remote stereogenic centers 3550 8, apart6 as well as conformational studies of biopolymers, e.g., ligand-receptor interactions. Snejana Novkova, Irena Philipova, and Blagoy Blagoev
TL;DR: The secondary structure of a 20-amino acid length synthetic peptide corresponding to the N terminus of the second subunit of hemagglutinin (HA2) of influenza virus A/PR8/34 and its interaction with phospholipid bilayers are investigated using ESR, Fourier transform infrared (FTIR), and CD spectroscopy.
TL;DR: The EC domain was shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity only sixfold lower than that of intact human BM-40, suggesting that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module.
TL;DR: It is concluded that neither the order of secondary structure elements, nor the preservation of any of the beta-hairpins present in this domain, is crucial for the ability of the polypeptide to fold, but they influence the folding and unfolding kinetics and could determine its folding pathway.
TL;DR: Observations indicate that association with negatively charged membranes induces a conformational change within α-lactalbumin to a flexible, molten globule-like state.
TL;DR: According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied.
Abstract: The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N4 4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 microM have a transition temperature (Tm) of 75.3 degrees C while at 0.5 microM the value drops to 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)-1 with a heat capacity change of 387 cal (K.mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The results support a view that the thermal unfolding of the molten globule states at pH 2 can be approximated by a two-state transition, but several results suggested that a combined mechanism incorporating the two- state transition and a gradual structural change would be more general in describing the conformational transition of the melting states.
TL;DR: The conformational analysis of the purified 33 kDa extrinsic polypeptide was carried out using FTIR spectroscopy with its self‐deconvolution and second derivative resolution enhancement as well as curve‐fitting procedures, and showed that the isolated polypePTide is characterized by a major proportion β‐sheet conformation.