Showing papers on "Circular dichroism published in 1994"
Book•
01 Apr 1994
TL;DR: Partial TABLE of CONTENTS: Circular Dichroism: An Introduction.
Abstract: PARTIAL TABLE OF CONTENTS: Circular Dichroism: An Introduction (G. Snatzke). From Pasteur to Parity Nonconservation: Theories of the Origin of Molecular Chirality (S. Mason). Theoretical Approach to Electronic Optical Activity (A. Koslowski, et al.). Vibrational Optical Acivity Theory (L. Nafie & T. Freedman). Fast Time-Resolved Circular Dichroism Measurements (D. Kliger & J. Lewis). The Octant Rule (D. Lightner). Circular Dichroism and Chirality of Dienes (J. Gawroski). Exciton Chirality Method: Principles and Applications (N. Berova & K. Nakanishi). A Model for How Polymers Amplify Chirality (M. Green). Induced CD or Polymers (E. Yashima & Y. Okamoto). Application of CD to the Study of Some Cholesteric Mesophase (G. Gottarelli & G. Spada). Circular Dichroism of Inorganic Complexes: Interpretation and Applications (R. Kuroda). Circular Dichroism of Peptides and Proteins (N. Sreerama & R. Woody). Peptide and Protein Conformational Studies with Vibrational Circular Dichroism and Related Spectroscopies (T. Keiderling). HPLC-CD: Stereochemical Analysis at Work (P. Salvadori, et al.). Applications of Chiroptical Spectroscopy in the Characterization of Compounds Having Pharmaceutical Importance (H. Brittain). Index.
1,404 citations
699 citations
TL;DR: Using circular dichroism spectroscopy it is shown that two complementary PNA strands can hybridize to one another to form a helical duplex and indicate that a (deoxy)ribose phosphate backbone is not an essential requirement for the formation of double helical DNA-like structures in solution.
Abstract: ALTHOUGH the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic acid (PNA)1-7 is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA7. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one another to form a helical duplex. There is a seeding of preferred chirality which is induced by the presence of an L- (or D-) lysine residue attached at the carboxy terminus of the PNA strand. These results indicate that a (deoxy)ribose phosphate backbone is not an essential requirement for the formation of double helical DNA-like structures in solution.
485 citations
TL;DR: It is concluded that the side chains in positions 7, 10, 12, 13, and 15 are directly involved in the receptor interaction while positions 28 and 29 are important for GLP-1 to adapt the conformation recognized by the receptor.
321 citations
TL;DR: Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation, and the observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the αR region over the β region, or that there is little preference for χ2 < 180° over χ 2 > 180°.
Abstract: It has often been assumed that the role of aromatic side chains in the far-ultraviolet region of protein circular dichroism (CD) is negligible. However, some proteins have positive CD bands in the 220-230 nm region which are almost certainly due to aromatic side chains. The contributions to the CD of interactions between tryptophan side chains and the nearest neighbor peptide groups have been studied, focusing on the indole Bb transition which occurs near 220 nm. Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation. Because of the low symmetry of indole, rotation about the C beta C gamma bond (dihedral angle chi 2) by 180 degrees generally leads to large changes in the CD, often causing the Bb band to reverse sign. When side-chain conformational preferences are taken into account, there is no strong bias for either positive or negative Bb rotational strengths. The observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the alpha R region over the beta region, or that there is little preference for chi 2 180 degrees. Nearest-neighbor-only calculations on individual tryptophans in 15 globular proteins also reveal a small bias toward positive Bb bands. Rotational strengths of the Bb transition for some conformations can be as large as approximately 1.0 Debye-Bohr magnetons in magnitude, corresponding to maximum molar ellipticities greater than 10(5) deg cm2/dmol. Although a substantial amount of cancellation occurs in most of the examples considered here, such CD contributions could be significant, especially in proteins of low helix content.
256 citations
TL;DR: The separation of PII fraction from the fraction of residues not assigned to alpha-helix or beta-sheet or -turns resulted in a distinctive PII CD spectrum and an unusual CD spectrum corresponding to the residual unassigned structures.
Abstract: A method to identify poly(L-proline)-type (PII) conformation in crystal structures of globular proteins is presented. Short segments of PII structure were identified in globular protein structures, and these form a significant fraction of the residues which are not assigned to alpha-helix, beta-sheet, and beta-turns. The fractions of alpha-helix, beta-sheet, beta-turns, PII, and unordered, identified in conjunction with the Kabsch and Sander method [(1983) Biopolymers 22, 2577], were incorporated in the analysis of circular dichroism (CD) spectra of proteins. The separation of PII fraction from the fraction of residues not assigned to alpha-helix or beta-sheet or -turns resulted in a distinctive PII CD spectrum and an unusual CD spectrum corresponding to the residual unassigned structures. The quality of prediction of PII fraction from CD spectra of proteins was comparable to that of beta-sheet and -turns.
236 citations
TL;DR: The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles.
Abstract: A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are consistent with a chain length dependent cooperative folding of the peptides into coiled coils.
232 citations
TL;DR: On the basis of the combined LD and CD evidence, it is proposed that the dye is loosely bound with poly(dA).poly(dT), probably in the major groove, and intercalated withPoly(dG).poly-dC, which each possess only one base-pair step.
Abstract: The modes of binding of the phenothiazinium dye methylene blue (1) to alternating and nonalternating polynucleotides and to calf thymus (CT) DNA have been characterized using linear dichroism (LD) and circular dichroism (CD) spectroscopy. With the polynucleotide [poly(dG-dC)](2) the interaction at low binding ratios is shown to be purely intercalative and the binding mode is insensitive to changes in ionic strength. The observed CD spectrum is bisignate, which may be due to intercalation at the different base-pair steps (5'G-C3' and 5'C-G3'), giving rise to CD signals of different sign and shape, By contrast, a single intercalative binding mode with the alternating AT polynucleotide [poly(dA-dT)](2) is likely only at very low ionic strength; at high ionic strength (200 mM phosphate, pH 6.9), a second binding mode is also manifest which is attributed to groove binding of the dye. The absorption and linear dichroism spectroscopic features of the methylene blue/CT-DNA (42% GC) complex reflect those of complexes of the dye with both [poly(dA-dT)](2) and [poly(dG-dC)](2); the circular dichroism spectrum of the methylene blue/CT-DNA complex and its variation with ionic strength reflect the complexity of even this simple system where numerous possible binding sites exist. Comparative binding to the nonalternating polynucleotides poly(dA).poly(dT) and poly(da).poly(dC), which each possess only one base-pair step, was also examined. On the basis of the combined LD and CD evidence, it is proposed that the dye is loosely bound with poly(dA).poly(dT), probably in the major groove, and intercalated with poly(dG).poly(dC).
230 citations
TL;DR: It is shown here using circular dichroism spectroscopy that RAP1 promotes the formation of one particular type of DNA quadruplex, parallel G4-DNA, which has implications for the possible use ofDNA quadruplexes in telomere-telomere association in vivo.
Abstract: Repressor-activator protein 1 (RAP1) has an essential role in the maintenance of yeast telomeres. Yeast telomeric DNA consists of simple repeated G-rich sequences that are bound by RAP1. We have found that RAP1, in addition to its known binding activity for double-stranded DNA, interacts with the G-rich strand containing guanine base (G)-tetrads. We show here using circular dichroism spectroscopy that RAP1 promotes the formation of one particular type of DNA quadruplex, parallel G4-DNA. Furthermore, RAP1 is able to bind to both preformed parallel and antiparallel DNA quadruplexes. These results have implications for the possible use of DNA quadruplexes in telomere-telomere association in vivo.
203 citations
TL;DR: While the neural network method performed slightly better than the other two methods at the basic level, the inclusion of the variable selection principle led to similar performance indices for all three methods.
198 citations
TL;DR: The structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.
Abstract: The application of linear and circular dichroism (LD and CD) in nucleic acid research id illustrated by recent results aimed at answering specific structural problem in the interaction of DNA with molecules of biological importance. We first consider the circumstances under which ligands, such as DAPI (4′, 6-diamidino-2-phenylindole), change their preferred binding mode in the minor groove to major groove binding or intercalation. As an extension of this problem we refer to the switch between groove binding and intercalation of structurally similar ligands such as ellipticines and trigonal ruthenium complexes. We also explore the use of LD and CD in the determination of the structure of the complex formed between the polynucleotide poly(dA) and the novel ‘peptide nucleic acid’, consisting of nucleic acid bases joined by a polyamide homomorphous with the deoxyribose-phosphate backbone of DNA. Finally, the structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.
TL;DR: A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > coTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants.
Abstract: Utilizing linear dichroism (LD), circular dichroism (CD), and fluorescence energy transfer, the binding geometries of a series of Co(3+)-porphyrins and their free ligands were examined. The compounds studied were Co-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) and its free ligand (H2-TMPyP), Co-meso-tetrakis(N-n-butylpyridinium-4-yl)porphyrin (CoTBPyP) and its free ligand (H2TBPyP), and Co-meso-tetrakis(N-n-octylpyridinium-4-yl)porphyrin (CoTOPyP). The two non-metalloporphyrins exhibit negative LD, having angles of roughly 75 degrees relative to the DNA helix axis. They also display negative CD and a significant contact energy transfer from the DNA bases. On the other hand, the three metalloporphyrins display orientation angles of roughly 45 degrees between the porphyrin plane and the helix axis of DNA. Furthermore, they exhibit positive CD and no contact energy transfer from DNA bases. These observations show that the metalloporphyrins are not intercalated whereas non-metalloporphyrins having four freely rotating meso-aryl groups intercalate between the base pairs of DNA. In the presence of KHSO5, the cobalt porphyrins cleave closed circular PM2 DNA in a single strand manner, i.e., a single activation event on the porphyrin leads to a break in one of the DNA strands. A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > CoTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants. Based on these and earlier observations, the metalloporphyrins appear bound to a partially melted region of DNA.
TL;DR: It is found that solvent is a very important factor in determining the secondary structure of an amino acid sequence in vitro and can override the propensity for a secondary structure due to sequence.
Abstract: We report here the effect of bulk solvent environment on the secondary structure of several peptides In previous work, equivocal peptide sequences that are predicted to be alpha-helical from amino acid preference but are found to be beta-strand in their proteins were shown to be alpha-helical in alcohol solvents and beta-strand in nonmicellar sodium dodecyl sulfate (SDS) by circular dichroism (CD) spectroscopy [Zhong, L, & Johnson, W C, Jr (1992) Proc Natl Acad Sci USA 89, 4462-4465] Here we show that equivocal sequences that are predicted to be beta-strand but are found to be alpha-helical follow the same pattern; they are alpha-helical in alcohol solvents and beta-strand in nonmicellar SDS Furthermore, we investigated a control sequence with only a strong alpha-helical propensity and a control sequence with only a strong beta-strand propensity Both of these well-behaved sequences followed the same pattern as the equivocal sequences The exceptionally stable Y(EAAAK)3A is an alpha-helix in all solvents, but analyses of the CD spectra indicate the loss of helix with an increase in beta-strand and other structures on changing solvent from trifluoroethanol (TFE) to SDS, similar to the other peptides We find that solvent is a very important factor in determining the secondary structure of an amino acid sequence in vitro and can override the propensity for a secondary structure due to sequence(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Findings strongly suggest that all the three sequences adopt an intramolecular i-motif structure for KRC6, a critical functional stretch of the c-ki-ras promoter proto-oncogene, besides the human telomeric sequence HTC4, may be suggestive of larger significance in the functioning of DNA.
Abstract: The structural behaviour of repetitive cytosine DNA is examined in the oligodeoxynucleotide sequences of (CCCTAA)3CCCT (HTC4), GC(TCCC)3TCCT(TCCC)3 (KRC6) and the methylated (CCCT)3TCCT(CCCT)3C (KRM6) by circular dichroism (CD), gel electrophoresis (PAGE), and ultra violet (UV) absorbance studies. All the three sequences exhibit a pH-induced cooperative structural transition as monitored by CD. An intense positive CD band around 285 nm develops on lowering the pH from 8 to slightly acidic condition, indicative of the formation of base pairs between protonated cytosines. The oligomers are found to melt in a fully reversible and cooperative fashion, with a melting temperature (Tm) of around 50 degrees C at pH 5.5. The melting temperatures are independent from DNA concentration, indicative of an intramolecular process involved in the structural formation. PAGE experiments performed with 32P-labeled samples as well as with normal staining procedures show a predominantly single band migration for all the three oligomers suggestive of a unimolecular structure. From pH titrations the number of protons required for generating the structures formed by HTC4, KRC6 and KRM6 results to be around six. These findings strongly suggest that all the three sequences adopt an intramolecular i-motif structure. The demonstration of i-motif structure for KRC6, a critical functional stretch of the c-ki-ras promoter proto-oncogene, besides the human telomeric sequence HTC4, may be suggestive of larger significance in the functioning of DNA.
TL;DR: Different spectra calculated between the wild-type spectra and those of mutants in which individual aromatic residues are replaced with non-chromophoric side chains show satisfactory agreement with experiment in most cases.
Abstract: We have applied exciton theory to estimate the circular dichroism (CD) contribution of the Trp Bb transition to the far-UV circular dichroism of globular proteins. Strong exciton couplets are predicted for a number of proteins, including dihydrofolate reductase (DHFR), chymotrypsin and chymotrypsinogen. These predicted CD spectra are dominated by the contributions of the closest pair, W47-W74 in DHFR and W174-W215 in chymotrypsinogen. The sign and magnitude of the predicted couplets are consistent with experimental data for DHFR and its W74L mutant, and with the previously unexplained CD changes upon chymotrypsin activation. More extensive coupled-oscillator interactions among all aromatic and peptide chromophores are described for DHFR and barnase. The total far-UV CD spectra predicted for these proteins agree poorly with experiment, primarily owing to difficulties in the calculation of the peptide CD. Nevertheless, difference spectra calculated between the wild-type spectra and those of mutants in which individual aromatic residues are replaced with non-chromophoric side chains show satisfactory agreement with experiment in most cases.
TL;DR: The results indicated that the favored chain orientation, that is, the major disulfide-bridged product formed under benign conditions, was the one that provides interchain electrostatic attractions between oppositely-charged amino acid residues in the e-g' and g-e' positions of the parallel coiled-coil and the g-g’ and e-e’ positions in the antiparallelcoil.
Abstract: The role of interchain electrostatic interactions in orientating alpha-helical chains to form two-stranded parallel and antiparallel coiled-coils has been investigated. Four disulfide-bridged coiled-coils were designed: parallel coiled-coils with interchain electrostatic attractions (P/A) and repulsions (P/R) and antiparallel coiled-coils with interchain electrostatic attractions (AP/A) and repulsions (AP/R). These coiled-coils were made by air oxidation of two 35-residue peptides with the appropriate heptad repeat (LaEbAcLdEeGfKg or LaAbEcLdKeGfEg) to give the desired interchain electrostatic interactions, and the appropriate position of the cysteine residue (C2 or C33) to give the desired chain orientation. The coiled-coils were characterized by circular dichroism spectroscopy, and their stabilities were assessed by guanidine hydrochloride and urea denaturations. The results indicated that the favored chain orientation, that is, the major disulfide-bridged product formed under benign conditions, was the one that provides interchain electrostatic attractions between oppositely-charged amino acid residues in the e-g' and g-e' positions of the parallel coiled-coil and the g-g' and e-e' positions in the antiparallel coiled-coil. When the electrostatic interactions were similar, the antiparallel coiled-coils were more stable than the parallel coiled-coils. However, the overall stability of the coiled-coils was either increased by interchain electrostatic attractions or decreased by interchain electrostatic repulsions, as determined by urea denaturation. Thus, the order of overall stability of these coiled-coils was AP/A > P/A > AP/R > P/R. This study demonstrates the importance of interchain electrostatic interactions in determining the parallel or antiparallel orientation of alpha-helical chains in two-stranded coiled-coils.
TL;DR: The role of lipids in the aggregation of three Alzheimer model peptides was investigated with circular dichroism spectroscopy and high-sensitivity titration calorimetry and the intrinsic binding constants were found to be constant and much smaller with K approximately 2-10 M-1.
Abstract: The role of lipids in the aggregation of three Alzheimer model peptides was investigated with circular dichroism spectroscopy and high-sensitivity titration calorimetry under conditions of low ionic strength. In solution, the peptides PAP(25-35)OH and @AP(25-35Nle)NH2 exhibit a reversible random- coil F! @-sheet (or @-structured aggregate) transition. Addition of lipid vesicles containing negatively charged lipids shifts the random-coil + @-sheet equilibrium almost completely toward @-sheet structure, which can be explained by the specific conditions created at the membrane surface: the cationic peptides are attracted to the negatively charged membrane, and the increase in peptide concentration together with the partial alignment of the peptide molecules then facilitates @-sheet formation. The third peptide, PAP- (25-35)NH2, also binds to the lipid membrane but was found to adopt an essentially random-coil structure, both with and without lipids. A quantitative characterization of the binding equilibrium was possible with high-sensitivity titration calorimetry. All three peptides exhibited exothermic binding enthalpies which varied between Lw= -2 kcal/mol for PAP(25-35)OH and-8 kcal/mol for BAP(25-35)NHz. The apparent binding constants, calculated with bulk concentrations, were large and varied between 500 and 5 X lo4 M-l, depending on the experimental conditions. However, after correction for electrostatic charge effects using the Gouy-Chapman theory, the intrinsic binding constants were found to be constant and much smaller with K - 2-10 M-l. The low intrinsic binding constants exclude significant hydrophobic interactions between the lipid membrane and the three Alzheimer model peptides even though residues 29-35 are considered to be part of a membrane-spanning domain in the full-length precursor protein. The predominance of electrostatic forces was demonstrated by addition of 0.1 M NaCl, which abolished the peptide-lipid interaction.
TL;DR: This chapter discusses the lipoproteins, structure, function, biosynthesis, and model for protein export of murein lipoprotein of Escherichia coli, the first integral membrane protein for which the primary structure was determined.
Abstract: Publisher Summary This chapter discusses the lipoproteins, structure, function, biosynthesis, and model for protein export. Murein lipoprotein of Escherichia coli was, apart from the basic myelin protein, the first integral membrane protein for which the primary structure was determined. It was the first protein in which a covalently bound lipid was demonstrated by determination of the structure of the lipid and its mode of attachment to the protein. The mature murein lipoprotein consists of 58 amino acid residues, in which no glycine, histidine, proline, phenylalanine, and tryptophan is present. The amino acid residues of murein lipoprotein are arranged such that all charged and hydrophilic side chains are located at one side, and all hydrophobic side chains at the opposing side of α-helix. Indeed, an α -helical content of over 80% was determined by circular dichroism measurements of lipoprotein samples isolated by using boiling sodium dodecyl sulfate, and can be done by employing a much milder procedure as well.
TL;DR: Comparison of the stability of the native and acidic molten globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride using the intact species and species modified by various degrees of acetylation of the lysyl epsilon-amino groups predicts that Gdn-HCl will produce substantially different effects on the conformational states of some proteins compared with those of urea.
TL;DR: The second-harmonic-generation (SHG) efficiency from a monolayer of oriented chiral molecules has a strong dependence upon the handedness of the circularly polarized excitation beam, which allows the study of chiral surfaces.
Abstract: The second-harmonic-generation (SHG) efficiency from a monolayer of oriented chiral molecules (R- or S-2,2'-dihydroxyl-1,1'binaphthyl) has a strong dependence upon the handedness of the circularly polarized excitation beam. The SHG circular-dichroism (CD) effect is much larger than in ordinary CD spectroscopy because SHG-CD is electric dipole allowed. The technique allows the study of chiral surfaces. The preference for left or right circularly polarized light is dependent on both the chirality (R or S) and the absolute orientation of the molecules (pointed up or down).
TL;DR: The secondary structure and the aggregation of fragment 25-35 of the beta-amyloid protein (beta AP(25-35)OH) under a variety of conditions are studied using circular dichroism spectroscopy.
Abstract: The beta-amyloid protein (39-43 amino acid residues) is the major constituent of the amyloid deposits found in brain of patients with Alzheimer's disease. Using circular dichroism spectroscopy, we have studied the secondary structure and the aggregation of fragment 25-35 of the beta-amyloid protein (beta AP(25-35)OH) under a variety of conditions. beta AP(25-35)OH in solution at pH 4.0 or 5.5 exhibits a concentration-dependent random coil beta-sheet transition. The equilibrium is characterized spectroscopically by an isodichroic point and can be described quantitatively by a simple association model with association constants between 1.8 x 10(4) M-1 (non-cooperative model, nucleation parameter sigma = 1) and 2.9 x 10(4) M-1 (cooperative model, sigma = 0.2). The enthalpy of association is delta H approximately -3 kcal/mol as determined by titration calorimetry. The equilibrium is shifted completely toward beta-structured fibrils at pH 7.4 where the Met-35 carboxyl group is fully charged. In contrast, removal of the charged carboxy terminus by amidation locks the equilibrium in the random coil conformation. Model calculations suggest an antiparallel beta-sheet structure involving residues 28-35 which is stabilized at both ends of the beta-sheet by ion pairs formed between Lys-28 and Met-35. Removal of fibrils via millipore filtration leads to solutions with random coil monomers only. Seeding these solutions with a few fibrils establishes a new random coil beta-sheet equilibrium.
TL;DR: The frequent occurrence of beta-sheet promoting residues such as Ile, Val, and Thr in the alpha-helical transmembrane segments of most integral membrane proteins suggests that the helical propensities of these residues are altered in the hydrophobic environment of the lipid bilayer.
Abstract: The frequent occurrence of beta-sheet promoting residues such as Ile, Val, and Thr in the alpha-helical transmembrane segments of most integral membrane proteins suggests that the helical propensities of these residues are altered in the hydrophobic environment of the lipid bilayer. Systematic studies of model peptides by circular dichroism spectroscopy in various micellar/vesicular media allow the establishment of a ranking order of helical propensity for uncharged amino acids in the membrane environment. In contrast to their conformational preferences in water, the helical proclivity of amino acids in membranes is shown to be governed by their side chain hydrophobicity, and by the hydropathy of the local peptide segments in which the residues reside.
TL;DR: The following approach was applied to the original data set to obtain an objective mathematical algorithm for the determination of a protein's tertiary structure class from its CD spectrum, which demonstrated well-separated all-beta class and denatured proteins.
TL;DR: The protein α-lactalbumin exists in a partially folded molten globule state at pH 2·0, the A state is believed to be compact, possessing a similar amount of secondary structure to the native state but having a flexible tertiary structure comprised mainly of non-specific hydrophobic clustering of residues.
TL;DR: The results demonstrate that PrP27-30 associates through a compact, metastable hydrophobic intermediate with an nonnative, nondenatured secondary structure and a tertiary structure close to the unfolded form, and propose a mechanism for scrapie amyloid association.
Abstract: The scrapie amyloid (prion) protein (PrP27-30) is a host-derived component of the infectious scrapie agent; the potential to replicate, propagate, and form amyloid is a result of the posttranslational event or conformational abnormality. In low concentrations of guanidine hydrochloride (Gdn.HCl), PrP27-30 dissociates into a compact equilibrium intermediate with a substantial portion of secondary structure, partially denatured tertiary structure, and tryptophan residues in an apolar environment [Safar, J., Roller, P. P., Gajdusek, D. C., & Gibbs, C. J., Jr. (1993) J. Biol. Chem. 27, 20276-20284]. Here we describe the characteristics of this metastable form as monitored by 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectroscopy and circular dichroism (CD) spectroscopy, and we propose a mechanism for scrapie amyloid association. The Gdn.HCl-induced equilibrium intermediate of PrP27-30 had multiple high-affinity hydrophobic binding sites for ANS, some close to the Trp residues. The amide CD spectrum of an acid-induced intermediate (A-form), in equilibrium at pH < 2.0, was similar to the Gdn.HCl-induced intermediate and suggested the presence of a significant portion of an alpha-helical or beta-turn secondary structure. In contrast, the PrP27-30 associated into aggregates in an all beta-sheet conformation with less ordered and more exposed hydrophobic side chains. The noncooperative unfolding of the Gdn.HCl-induced intermediate at high temperature was irreversible and correlated with the loss of infectivity. The results demonstrate that PrP27-30 associates through a compact, metastable hydrophobic intermediate with an nonnative, nondenatured secondary structure and a tertiary structure close to the unfolded form.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The binding study of PR-39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane-bound, and the question of the possible mechanism of interaction ofPR-39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane is addressed.
Abstract: PR-39 is a 4719-Da peptide isolated from pig intestine and belonging to the recently discovered family of Pro+Arg-rich antibacterial peptides. PR-39 does not lyse Escherichia coli, instead the lethal action is probably linked to the termination of DNA and protein synthesis [Boman, H. G., Agerberth, B. & Boman, A. (1993) Infect. Immun. 61, 2978-2984]. Circular dichroism and Fourier-transform infrared spectroscopy have been used to investigate the secondary structure of PR-39 in the absence or presence of lipids. According to the circular dichroic data, this structure is not altered upon incubation of PR-39 with negatively charged vesicles, although the infrared spectra suggest that the hydrogen bond pattern is modified upon the peptide-lipid interaction. This is detected by a shift in the maximum wavelength of absorption of PR-39 from 1636 cm-1 in the absence of lipids to 1645 cm-1 in the presence of lipids. We have further addressed the question of the possible mechanism of interaction of PR-39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane. PR-39 induced a calcein release from large unilamellar vesicles, which is dependent upon the peptide concentration and upon the presence of negatively charged lipid (glycerophosphoglycerol) in the membrane. The binding study of PR-39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane-bound. Addition of PR-39 to a planar lipid bilayer induced a linear increase in the current but no channel formation was observed since no discrete steps of conductance occurred.
TL;DR: The constitutive NOS peptide binds to CaM in a calcium-dependent manner with 1:1 stoichiometry as determined by polyacrylamide gel electrophoresis of the peptide-CaM complex in 4 M urea and can adopt an alpha-helical structure which bears close resemblance to the structure of the CaM-bound form of the myosin light chain kinases.
TL;DR: The results suggest that stacking inhibits protonation of the DNA-bound porphyrin, which is consistent with a model in which the interacting TBOPP in the less stacked form has a different relative orientation than the more stacked form.
Abstract: The interactions between the water-soluble tentacle porphyrin meso-tetrakis[4-[(3-(trimethylammonio)propyl)oxy]phenyl]porphine (TBOPP) and calf thymus (CT) DNA had been found to exhibit unusual features: (a) At pH 7.0, TBOPP underwent extensive self-stacking along the DNA surface (Marzilli, et al. J . Am. Chem. SOC. 1992, 114, 7575); (b) the DNA-bound form of TBOPP was extensively protonated even at pH 7.0 (Petho, et al. J . Chem. Soc., Chem. Commun. 1993, 1547). In the studies presented here, the formation of the TBOPP-DNA adduct was investigated under various conditions of pH, salt concentration, [porphyrin]/[DNA base pair] ratio (R) , and absence/presence of buffer. Under all conditions studied, TBOPP was an outside binder. Methods employed included circular dichroism (CD), UV-visible absorbance, and fluorescence spectrophotometry. Timedependence experiments were also conducted in order to detect any change in binding characteristics over time. Evidence for outside-bound, self-stacked forms of TBOPP included a large conservative induced visible CD feature, whereas the evidence for DNA-bound, protonated TBOPP included a Soret band red-shifted to 451 nm, a single positive induced CD band at 45 1 nm, and a red-shifted fluorescence band at 740 nm. At least two forms of the outside-bound, self-stacked porphyrin were evident. At high R (0.25), hypochromicity of the Soret band and a conservative type CD feature indicated that TBOPP was extensively stacked. At lower R (0.05), increases in the Soret band intensity and changes in the band shape suggested a less extensively stacked form, but this form of TBOPP had a larger conservative feature in the CD spectrum. The intensity of the conservative band suggested that the less stacked form is very stable at R = 0.05. The CD data are consistent with a model in which the interacting TBOPP in the less stacked form has a different relative orientation than the more stacked form. Both high salt concentration and PIPES buffer appeared to stabilize the outside-bound, self-stacked form of TBOPP. At R = 0.01, a decrease in CD band intensity suggested that the porphyrin was less stacked than at R = 0.05; this change in stacking occurred over 1-2 h. Under these low R conditions where outside-bound self-stacking is less favored, the degree of TBOPP protonation is highest. The conservative-type CD spectrum changed to a simple positive CD band upon protonation of TBOPP, indicating that the binding mode underwent a transition from outside binding with self-stacking to simple outside binding. High salt concentration (100 mh4 NaCl) was found to decrease the degree of protonation, and the presence of PIPES buffer also decreased the degree of protonation at pH 7.0. Under conditions which favored protonation (low salt or no PIPES), the degree of protonation was found to increase gradually during the first few hours after sample preparation. Since in the time course of the experiment the total contents of the solution were unchanged, and since the isolated unstacked porphyrin should protonate quickly, these results suggest that stacking inhibits protonation of the DNA-bound porphyrin.
TL;DR: The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods and it is shown that at least one of the three tryptophan residues in the A form is accessible to water.
Abstract: The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.
TL;DR: The results suggest that phosphatidyl glycerol not only is involved in trimer formation but also has a structural role within the monomers.
Abstract: A detailed comparison has been made between dichroic steady-state spectroscopic properties at 77 K of several trimeric and monomeric forms of the major chlorophyll a/b binding protein (LHC-II) from pea. Monomeric forms were obtained by applying high concentrations of nonionic detergents, by a lipase treatment, or by a chymotrypsin/trypsin treatment. The latter treatments removed phosphatidyl glycerol essential for trimer formation. The absorption and dichroism spectra indicate that for trimeric LHC-II the chlorophyll b absorption region is centered around 649 nm and is composed of at least five subbands near 640, 647, 649, 652, and 656 nm. The chlorophyll a absorption region is centered around 670 nm and is composed of at least five bands near 661, 668, 671, 673, and 676 nm. The chlorophyll b band near 647 and 652 nm and the chlorophyll a bands near 668 and 673 nm are absent in the circular dichroism spectrum after monomerization. A configuration in which pigments of the same nature located on different monomers become excitonically coupled in the trimer could explain these results. In monomers obtained in high concentrations of nonionic detergents, no additional bands have disappeared, but the absorption spectra of the other two types of monomers lack the bands at 640 and 661 nm. These monomers have lost some chlorophyll a and b according to the fluorescence emission spectra, which show contributions from free chlorophyll a and b. The results suggest that phosphatidyl glycerol not only is involved in trimer formation but also has a structural role within the monomers.