Showing papers on "Circular dichroism published in 1997"

Circular Dichroism and Linear Dichroism

Abstract: 1. Spectroscopy, chirality, and oriented systems 2. Circular dichroism of biomolecules 3. Linear dichroism of biomolecules 4. Linear dichroism of small molecules 5. Analysis of circular dichroism: electric dipole allowed transitions 6. Analysis of circular dichroism: magnetic dipole allowed transitions and magnetic CD 7. Circular dichroism formalism A1. Vectors A2. Determination of equilibrium binding constants A3. Momentum-dipole equivalence

Chirality Assignment of Amines and Amino Alcohols Based on Circular Dichroism Induced by Helix Formation of a Stereoregular Poly((4-carboxyphenyl)acetylene) through Acid−Base Complexation

Abstract: An optically inactive polyacetylene, poly((4-carboxyphenyl)acetylene) (poly-1), exhibits an induced circular dichroism (ICD) in the UV−visible region upon complexation with chiral amines and amino alcohols in DMSO and in the film, the sign of which reflects the stereochemistry including bulkiness, type (primary, secondary, or tertiary), and absolute configuration of the amines. Therefore, the polyacetylene can be used as a novel probe for determining the chirality of amines. Most primary amines and amino alcohols of the same configuration gave the same sign for the induced Cotton effect; however, secondary and/or tertiary amines used in the present study tended to show Cotton effect signs opposite to those of the primary amines and amino alcohols of the same configuration. The magnitude of the ICD likely increases with an increase in the bulkiness of the chiral amines. The complexation dynamics during the formation of the helical structure of poly-1 with chiral amines were investigated on the basis of the...

Interaction of Alzheimer beta-amyloid peptide(1-40) with lipid membranes.

Abstract: The β-amyloid peptide βAP(1−40), a 40-amino acid residues peptide, is one of the major components of Alzheimer's amyloid deposits. βAP(1−40) exhibits only a limited solubility in aqueous solution and undergoes a concentration-dependent, cooperative random coil ⇄ β-structure transition for Cpep > 10 μM [Terzi, E., Holzemann, G., and Seelig, J. (1995) J. Mol. Biol. 252, 633−642]. In the presence of acidic lipid, the equilibrium is shifted further toward β-structured aggregates. We have now characterized the lipid−peptide interaction using circular dichroism (CD) spectroscopy, lipid monolayers, and deuterium and phosphorus-31 solid-state nuclear magnetic resonance (NMR). CD spectroscopy revealed a distinct interaction between βAP(1−40) and negatively charged unilamellar vesicles. In addition to the random coil ⇄ β-structured aggregate equilibrium at low lipid-to-peptide (L/P) ratios, a β-structure → α-helix transition was observed at L/P > 55. βAP(1−40) was found to insert into acidic monolayers provided the...

Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis.

Abstract: Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Automated design of the surface positions of protein helices.

Abstract: Using a protein design algorithm that quantitatively considers side-chain interactions, the design of surface residues of alpha helices was examined. Three scoring functions were tested: a hydrogen-bond potential, a hydrogen-bond potential in conjunction with a penalty for uncompensated burial of polar hydrogens, and a hydrogen-bond potential in combination with helix propensity. The solvent exposed residues of a homodimeric coiled coil based on GCN4-p1 were designed by using the Dead-End Elimination Theorem to find the optimal amino acid sequence for each scoring function. The corresponding peptides were synthesized and characterized by circular dichroism spectroscopy and size exclusion chromatography. The designed peptides were dimeric and nearly 100% helical at 1 degree C, with melting temperatures from 69-72 degrees C, over 12 degrees C higher than GCN4-p1, whereas a random hydrophilic sequence at the surface positions produced a peptide that melted at 15 degrees C. Analysis of the designed sequences suggests that helix propensity is the key factor in sequence design for surface helical positions.

Structure, Stability, and Activity of Adsorbed Enzymes

Abstract: A proteolytic enzyme, α-chymotrypsin, and a lipolytic enzyme, cutinase, were adsorbed from aqueous solution onto a hydrophobic Teflon surface and a hydrophilic silica surface. We investigated the influence of adsorption on the structure, the structure thermal stability and the activity of these enzymes. Probing the protein structure by circular dichroism spectroscopy indicates that Teflon promotes the formation of helical structure in α-chymotrypsin, but the reverse effect is found with cutinase. The perturbed protein structures on Teflon are remarkably stable, showing no heat-induced structural transitions up to 100°C, as monitored by differential scanning calorimetry. Contact with the hydrophilic silica surface leads to a loss in the helix content of both proteins. Differential scanning calorimetry points to a heterogeneous population of adsorbed protein molecules with respect to their conformational states. The fraction of the native-like conformation in the adsorbed layer increases with increasing coverage of the silica surface by the proteins. The specific enzymatic activity in the adsorbed state qualitatively correlates with the fraction of proteins in the native-like conformation.

Peptide hydrophobicity controls the activity and selectivity of magainin-2 amide in interaction with membranes

Abstract: The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an α-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic ...

Acid-Induced Unfolding of Cytochrome c at Different Methanol Concentrations: Electrospray Ionization Mass Spectrometry Specifically Monitors Changes in the Tertiary Structure†

Abstract: The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the α-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the a...

A method for assessing the stability of a membrane protein.

Abstract: The integral membrane protein diacylglycerol kinase (DGK) from Escherichia coli has been reversibly unfolded in a protein/detergent/mixed micelle system by varying the molar ratio of n-decyl beta-D-maltoside (DM) and sodium dodecyl sulfate (SDS). Unfolding was monitored by circular dichroism (CD) and ultraviolet (UV) absorbance spectroscopy. When unfolding is monitored by measuring changes in absorbance at 294 nm, two distinct denaturation phases are observed, indicative of a stable intermediate. When CD is used as a conformational probe, the resulting denaturation curve contains only one major transition, which corresponds to the first unfolding phase observed by absorbance changes. The unfolding behavior of several mutant proteins in which the tryptophan residues were selectively replaced made it possible to assign the first unfolding phase to a denaturation event in a cytoplasmic domain and the second phase to denaturation of the membrane-embedded portion of the protein. The denaturation curves fit well to a model which assumes two cooperative transitions and a linear relationship between unfolding free energy and SDS concentration. Extrapolation back to zero denaturant indicates an unfolding free energy of 6 kcal/mol for the cytoplasmic domain and 16 kcal/mol for the transmembrane domain. The high apparent stability of the transmembrane domain could explain the high degree of tolerance to amino acid substitutions seen for DGK and other membrane proteins. The approach described in this paper may be applicable to other membrane protein systems.

Characterization of the interactions of Alzheimer beta-amyloid peptides with phospholipid membranes.

Abstract: Increasing evidence suggests that Alzheimer beta-amyloid peptides (AAPbeta) may be toxic agents in Alzheimer disease. We investigated the possibility that the toxicity may be the result of peptide-lipid interactions, involving either the cell membrane or the intracellular vesicular system. The interaction of the AAPbeta-(1-40), AAPbeta-(1-42), AAPbeta-(9-25) and AAPbeta-(25-35)-peptides with acidic and zwitterionic phospholipids was investigated by means of circular dichroism, vesicle disruption and lipid-aggregation assays. These studies were undertaken at peptide concentrations approaching in vivo levels and at physiological salt concentrations. Circular-dichroism studies demonstrate that acidic phospholipids induce a conformational change from random coil to beta structure in AAPbeta-(1-40)-peptide and AAPbeta-(1-42)-peptide at pH 6.0. In contrast, at pH 7.0, only AAPbeta-(1-42)-peptide was induced to adopt beta structure. Phosphatidylinositol was the most efficient inducer of beta structure in AAPbeta-(1-42)-peptide. To further investigate the peptide-lipid interactions, we examined the ability of the AAPbeta peptides to disrupt and/or aggregate phospholipid vesicles. These properties were found to be mediated predominantly through electrostatic interactions with the phospholipid headgroup. The data presented in this paper have implications for AAPbeta toxicity and senile-plaque formation.

Folding dynamics of the src sh3 domain

Abstract: The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure Kinetic studies confirmed the two-state character of the folding reaction Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (04−37 M) Estimates of the free energy of unfold

Fluorescent Chemosensor for Organic Guests and Copper(II) Ion Based on Dansyldiethylenetriamine-Modified β-Cyclodextrin

Abstract: A modified cyclodextrin containing a dansyldiethylenetriamine metal-binding group (6-deoxy-6-N-(N‘‘-dansyldiethylenetriamino)-β-cyclodextrin, CD-dien-DNS) was synthesized. The conformation of CD-dien-DNS was studied by 2D NMR (ROESY spectra) in D2O, by circular dichroism, and by fluorescence. The results were compared with those previously obtained with the analogous 6-deoxy-6-N-(N‘-dansylethylenediamino)-β-cyclodextrin (CD-en-DNS) and were consistent with the self-inclusion of the dansyl group within the macrocycle cavity. However, the orientation of the dansyl group for CD-dien-DNS was found to be equatorial, whereas for CD-en-DNS it was axial, suggesting a dependence of the orientation of the dansyl group upon the length of the linker. In the presence of lipophilic organic molecules, CD-dien-DNS showed sensing properties similar to those observed for CD-en-DNS, suggesting a similar “in-out” movement of the dansyl group, due to competitive inclusion of the guest. Unlike CD-en-DNS, CD-dien-DNS was found ...

Direct SCF direct static-exchange calculations of electronic spectra

Abstract: Applications of the direct SCF direct static-exchange method are reviewed for three different types of systems (free molecules, polymers, and surface adsorbates) and for six types of spectroscopy (X-ray absorption-, -emission-, and -shake-up spectroscopy, ultraviolet photoelectron emission, X-ray Raman and circular dichroism spectroscopy.

Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity.

Abstract: The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 °C, the addition of Mg2+ (Kd ∼ 50 μM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] ∼ 0.8 mM, Hill coefficient ∼ 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; ∼30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.

Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria.

Abstract: The first three-dimensional structure of a type IIa bacteriocin from lactic acid bacteria is reported. Complete 1H resonance assignments of leucocin A, a 37 amino acid antimicrobial peptide isolated from the lactic acid bacterium Leuconostoc gelidum UAL187, were determined in 90% trifluoroethanol (TFE)−water and in aqueous dodecylphosphocholine (DPC) micelles (1:40 ratio of leucocin A:DPC) using two-dimensional NMR techniques (e.g., DQF-COSY, TOCSY, NOESY). Circular dichroism spectra, NMR chemical shift indices, amide hydrogen exchange rates, and long-range nuclear Overhauser effects indicate that leucocin A adopts a reasonably well defined structure in both TFE and DPC micelle environments but exists as a random coil in water or aqueous DMSO. Distance geometry and simulated annealing calculations were employed to generate structures for leucocin A in both lipophilic media. While some differences were noted between the structures calculated for the two different solvent systems, in both, the region encomp...

Kinetics of folding of the IgG binding domain of peptostreptoccocal protein L

Abstract: The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding...

Engineering of peptide β-sheet nanotapes

Abstract: A set of principles are outlined for the design of short oligopeptides which will self-assemble in appropriate solvents into long, semi-flexible, polymericβ-sheet nanotapes. Their validity is demonstrated by experimental studies of an 11-residue peptide (DN1) which forms nanotapes in water, and a 24-residue peptide (K24) which forms nanotapes in non-aqueous solvents such as methanol. Circular dichroism (CD) spectroscopy studies of the self-assembly behaviour in very dilute solutions (µm) reveal a simple transition from a random coil-to-β-sheet conformation in the case of DN1, but a more complex situation for K24. Association of DN1 is very weak up to a concentration of 40 µm at which there is a sudden increase in the fraction of peptide in the β-sheet structure, indicative of an apparent ‘critical tape concentration’. This is shown to arise from a two-step self-assembly process: the first step being a transition from a random coil to an extended β-strand conformation, and the second the addition of this β-strand to a growing β-sheet. Both peptides are shown to gel their solvents at concentrations above 2×10 -3 volume fraction: these gels are stable up to the boiling point of the solvents. Rheology measurements on gels of the 24-residue peptide in 2-chloroethanol reveal that the tapes form an entangled network with a mesh size of 10–100 nm for peptide volume fractions 0.03–0.003; the persistence length of the tape is 13 nm or greater, indicative of a moderately rigid polymer; the tapes are about a single molecule in thickness. The mechanical properties of the gels in many respects are comparable to those of natural biopolymers such as gelatin, actin, amylose and agarose.

Comparison of nh exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides

Abstract: Circular dichroism and NH exchange are compared directly as techniques for measuring helix content in peptides and the parameters of the helix-coil transition. To cover a broad range of helix contents, alanine-based peptides with chain lengths varying from 12 to 22 residues are examined over the temperature range from 0.6 to 26.9 degrees C in 1 M sodium chloride, 2H2O. Helix-coil transition theory independently fits both circular dichroism and exchange data, but the helix contents measured by exchange are larger than those measured by circular dichroism. The two techniques are brought into agreement by removing the assumption that the intrinsic chemical exchange rate in the helix is the same as the exchange rate measured for short unstructured model peptides. This modification allows the circular dichroism and NH exchange data to be described by the same set of helix parameters and indicates that the intrinsic exchange rate in the presence of helical structure is reduced approximately 17% relative to the rates measured in unstructured models. To test the possibility that this effect is electrostatic in origin, the sensitivity of the exchange reaction to ionic strength is determined. A substantial dependence of exchange rate on ionic strength is found, but the form of the dependence is complex. In studies of the exchange rates of native proteins, the exchange-competent form of the protein is assumed to exchange with the same rate constant as a blocked dipeptide with the identical amino acid sequences. Our result suggests that this assumption will be seriously in error in some cases because of charge effects in the protein.

Structural and kinetic description of cytochrome c unfolding induced by the interaction with lipid vesicles.

Abstract: The interaction of cytochrome c with anionic lipid vesicles of DOPS induces an extensive disruption of the native structure of the protein. The kinetics of this lipid-induced unfolding process were investigated in a series of fluorescence- and absorbance-detected stopped-flow measurements. The results show that the tightly packed native structure of cytochrome c is disrupted at a rate of approximately 1.5 s-1 (independent of protein and lipid concentration), leading to the formation of a lipid-inserted denatured state (DL). Comparison with the expected rate of unfolding in solution (approximately 2 x 10(-3) s-1 at pH 5.0 in the absence of denaturant) suggests that the lipid environment dramatically accelerates the structural unfolding process of cytochrome c. We propose that this acceleration is in part due to the low effective pH in the vicinity of the lipid headgroups. This hypothesis was tested by comparative kinetic measurements of acid unfolding of cytochrome c in solution. Our absorbance and fluorescence kinetic data, combined with a well-characterized mechanism for folding/unfolding of cytochrome c in solution, allow us to propose a kinetic mechanism for cytochrome c unfolding at the membrane surface. Binding of native cytochrome c in water (NW) to DOPS vesicles is driven by the electrostatic interaction between positively charged residues in the protein and the negatively charged lipid headgroups on the membrane surface. This binding step occurs within the dead time of the stopped-flow experiments ( DL) is limited by the rate of disruption of the hydrophobic core in MS. Equilibrium spectroscopic measurements by near-IR and Soret absorbance, fluorescence, and circular dichroism showed that DL has native-like helical secondary structure, but shows no evidence for specific tertiary interactions. This lipid-denatured equilibrium state (DL) is clearly more extensively unfolded than the A-state in solution, but is distinct from the unfolded protein in water (UW), which has no stable secondary structure.

Organophosphorus Hydrolase Is a Remarkably Stable Enzyme That Unfolds through a Homodimeric Intermediate

Abstract: Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.

Synthesis and Studies on the Effect of 2-Thiouridine and 4-Thiouridine on Sugar Conformation and RNA Duplex Stability

Abstract: In order to understand the effect of 2-thiouridine (s2U) substitution on RNA structure and the potential for stabilization of tRNA codon-anticodon interactions through s2U-34 modification, a pentamer RNA sequence, Gs2UUUC, was synthesized and characterized by NMR spectroscopy. The single strand contains the UUU anticodon sequence of tRNALys with flanking GCs to increase duplex stability. Regiochemical effects of uridine thiolation were determined by comparing the structure and stability of the 2-thiouridine containing oligonucleotide with an identical sequence containing 4-thiouridine (s4U) and also the normal uridine nucleoside. Circular dichroism spectrum indicated an A-form helical conformation for Gs2UUUC which was further confirmed by 2D ROESY NMR experiments. The duplex stability of the three pentamers complexed with a 2'-O-methyl-ribonucleotide complementary strand, GmAmAmAmCm, was determined by UV thermal melting studies and by 1H NMR spectroscopy. The duplex containing s2U has a T m of 30.7 degrees C compared to 19. 0 degrees C for the unmodified control and 14.5 degrees C for the s4U containing duplex. The results from UV experiments were corroborated by imino proton NMR studies that show proton exchange rates, chemical shift differences, and NH proton linewidths indicative of the stability order s2U >U >s4U. The magnitude of the effect of s2U in our model system is comparable to the 20 degrees C stabilization observed by Grosjean and co-workers for 2-thiolation in a codon-anticodon model system composed of two tRNAs with complementary anticodon sequences [Houssier, C., Degee, P., Nicoghosian, K. and Grosjean, H. (1988) J. Biomol. Struct. Dyn., 5, 1259-1266].