Showing papers on "Circular dichroism published in 1986"

Calculation of protein conformation from circular dichroism.

Abstract: Publisher Summary This chapter discusses some recent developments in the estimation of various conformations in a protein molecule from its circular dichroism (CD) spectrum in the ultraviolet region Optical rotatory dispersion (ORD) and CD are two chiroptical phenomena, which differentiate two enantiomers They are caused by different interactions of left- and right-circularly polarized light with chiral molecules Chirality is a geometric property of molecules; the corresponding substances are therefore optically active At present CD has replaced ORD for the conformational analysis of proteins because CD bands that are characteristic of various secondary structures can be directly observed The very simplicity of the CD method and the short time required for the measurements are extremely attractive It is often advantageous to compare the CD analysis with empirical predictions of secondary structure of proteins from sequences, if such are available All current methods for CD analysis are developed to determine not only the helix and β-form but also the β-turn and unordered forms

Stabilization of the ribonuclease S‐peptide α‐helix by trifluoroethanol

Abstract: The effects of trifluoroethanol (TFE) on the stability of the alpha-helix formed by ribonuclease S-peptide, residues 1-19 of ribonuclease A, were studied by measuring circular dichroism as a function of TFE concentration, pH, and temperature. The S-peptide forms an unusually stable alpha-helix, which is known to be stabilized by TFE. The magnitude of the effect of charged groups on the peptide, manifested by the change in alpha-helix stability as a function of pH, was not altered significantly by either TFE concentration or temperature, indicating that the lower dielectric constant of TFE is not important in the stabilization of this alpha-helix. This suggests that the alpha-helix might be stabilized by many interactions in addition to the effects of charges. The titration curve of circular dichroism vs. TFE concentration appears to be cooperative at 0 degree C, but becomes progressively less cooperative at temperatures between 25 and 75 degrees C. The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.

Conformations of Signal Peptides Induced by Lipids Suggest Initial Steps in Protein Export

Abstract: Despite the requirement for a functional signal sequence in protein export, little is known of the conformational properties and membrane interactions of these highly hydrophobic amino terminal extensions on nearly all exported proteins. The Escherichia coli lambda phage receptor signal sequence was studied in phospholipid monolayers by circular dichroism and Fourier transform infrared spectroscopy; the signal peptide was shown to prefer an alpha-helical conformation when inserted into the lipid phase. However, interaction with the lipid surface without insertion induced the signal sequence, which is unstructured in bulk aqueous solution, to adopt a beta structure. These observations are combined in a model for the initial steps in signal sequence-membrane interaction in vivo.

Optical properties of polymers

Abstract: Well, someone can decide by themselves what they want to do and need to do but sometimes, that kind of person will need some optical properties of polymers references. People with open minded will always try to seek for the new things and information from many sources. On the contrary, people with closed mind will always think that they can do it by their principals. So, what kind of person are you?

Theory of the interaction of light with large inhomogeneous molecular aggregates. II. Psi‐type circular dichroism

Abstract: A theory of the polymer and salt induced (psi)‐type circular dichroism observed in DNA aggregates is presented. Using the main formalism developed in the previous paper to treat the interaction of light and large, dense molecular aggregates, it is shown that the anomalously large signals observed in the circular dichroism of certain molecular aggregates result from: (a) the presence of a long‐range chiral structure in the aggregate; (b) delocalization throughout the entire particle of the light‐induced excitations in the chromophores. This delocalization and the resulting ‘‘collective response’’ of the chromophores in the aggregate is favored in particles having a three‐dimensional packing. It is shown that to describe adequately the internal field in these aggregates, intermediate and radiation coupling mechanisms should be taken into account in addition to the regular dipole–dipole interactions. Furthermore, no dipole approximation in the exponentials of the form eik⋅x are made. It is shown that in these circumstances, one of the circular polarizations of the light can exchange energy more efficiently than the opposite polarization. This gives rise to a circular dichroism signal whose magnitude is proportional to the overall size and long‐range chiral nature of the aggregates. The theory is applied to two cases: (1) to the dimer ApA when it is shown (for the case of this small system) to reduce to the classical theories of DeVoe and Tinoco, and (2) for a toroidal aggregate of DNA of 3000 A diameter with an internal chiral structure, as found by Haynes et al. in polylysine–DNA condensates. Good qualitative agreement with the observed spectra is found. The theory represents the first successful attempt to explain the physical origin of the psi‐type CD effect. Useful information regarding the chiral structure of the aggregates can be inferred from the theory.

Preferential conformation of substance P in solution.

Abstract: The three-dimensional structure of substance P has been studied by 1H-NMR, (500 MHz), and by circular dichroism (CD) in different solvents. The analysis of the different NMR parameters suggest that substance P adopts a rather extended structure in dimethylsulfoxide and pyridine. In water, besides the aggregation phenomenon, the monomeric substance P presents a complex conformational equilibrium. The addition of sodium dodecylsulfate to the aqueous solution induces, as shown by CD spectroscopy, a preferential alpha-helical conformation. And in methanol three structural conclusions may be drawn: the flexibility of the N-terminal Arg-Pro-Lys, the alpha-helical structure of Pro4-Gln5-Gln6-Phe7-Phe8 and the interaction of the C-terminal carboxamide with the primary amides from both glutamines.

Structure-Activity Studies of Interleukin-2

Abstract: The critical role of interleukin-2 (IL-2) in immune response heightens the need to know its structure in order to understand its activity. New computer-assisted predictive methods for the assignment of secondary structure together with a method to predict the tertiary structure of a protein from data on its primary sequence and secondary structure were applied to IL-2. This method generated four topological families of structures, of which the most plausible is a right-handed fourfold alpha-helical bundle. Members of this family were shown to be compatible with existing structural data on disulfide bridges and monoclonal antibody binding for IL-2. Experimental estimates of secondary structure from circular dichroism and site-directed mutagenesis data support the model. A region likely to be important in IL-2 binding to its receptor was identified as residues Leu36, Met38, Leu40, Phe42, Phe44, and Met46.

Folding of dihydrofolate reductase from Escherichia coli

Abstract: The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Abstract: Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D2O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.

Organization of Carotenoid-Phospholipid Bilayer Systems. Incorporation of Zeaxanthin, Astaxanthin, and their C50 Homologues into Dimyristoylphosphatidylcholine Vesicles

Abstract: Four carotenoids (zeaxanthin 1, astaxanthin 2, and their C50 synthetic isoprene-homologues 3 und 4) have been incorporated into bilayer unilamellar vescles of dimyristoylphospatidylcholine as proved by coelution on a Sepharose column. The incorporation into the bilayer proper has been proved by the sensitivity to phase transition of UV/VIS spectra and circular dichroism. The UV/VIS absorptions of the carotenoids are typical of a lipidic environment. At the temperature of phase transition occur both intermolecular phenomena (aggregation of carotenoid molecules) and intramolecular changes (conformational change). The relative solubilities of the various carotenoids in this phospholipid membrane can be fitted with molecular parameters (length of the lipophilic carotenoid segment vs. lipid bilayer thickness).

Nitrosyliron(III) hemoglobin: autoreduction and spectroscopy

Abstract: Nitrosyl complexes of the iron(III) forms of myoglobin, human hemoglobin, Glycera dibranchiata hemoglobins (Hbm and Hbh), and model iron(II) and iron(III) synthetic porphyrins including octaethylporphyrin (OEP) have been prepared. The iron(III) heme proteins are electron spin (paramagnetic) resonance (ESR) silent, while hexacoordinate solution structures are indicated for [Fe(OEP)(NO)2]ClO4 and for Hbm(II)NO, which has an ESR spectrum similar to that of Mb(II)NO and the hexacoordinate iron(II) model complex Fe(OEP)NO(BzIm). The splitting of the alpha- and beta-bands in the optical spectrum of Mb(III)NO and Hbh(III)NO contrasts markedly with the sharp, single bands observed in that of Hbm-(III)NO. The nondegeneracy of the dxz and dyz orbitals in Mb(III)NO and Hbh(III)NO is attributed to the influence of the distal histidine. Circular dichroism spectra were obtained for Hbm(III)NO, Hbm(II)NO, Hbh(III)NO, Hbh(II)NO, Mb(II)NO, and Mb(III)NO. The vicinal chiral center contribution that governs the heme protein CD leads to low Kuhn anisotropies, which have been used to assign certain electronic transitions. The Hb(III)NO spectrum is not stable but transforms into that of Hb(II)NO. This autoredox process follows kinetics that are first order in FeIIINO. The relative rates of autoreduction (25 degrees C, 1 atm NO) are Mb(III)NO less than Hbm(III)NO less than Hb alpha(III)NO less than HbA(III)NO. At high NO partial pressure or after "recycling" of HbA, the rates of reduction decrease. The first step in the reaction of NO with the ferric heme is the reversible formation of the formally iron(III) adduct. This reacts with another molecule of NO, generating the final heme(II)-NO via nitrosylation of NO itself or of an endogenous nucleophile. Kinetic and spectroscopic evidence shows involvement of trans-heme-(NO)2 in the reaction. The activation parameters delta H and delta S were determined. The overall reaction is photoenhanced.

The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study

Abstract: Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.

Physicochemical characterization of the heat-stable microtubule-associated protein MAP2.

Abstract: MAP2 purified without heat treatment was compared to MAP2 prepared with the usual boiling step. No significant differences were found by isoelectric focusing, peptide mapping, fluorescence spectroscopy and circular dichroism. Hydrodynamically, MAP2 is a monomer with s°20,w, = 3.5 ± 0.1 S and Mr= 220 000. The molecular mass was measured by sedimentation equilibrium and verified by gel chromatography in denaturing solvent and dodecyl sulfate gel electrophoresis at different acrylamide concentrations. The high frictional ratio, f/fmin= 3.7, indicated that MAP2 was clearly not globular but had either a very elongated shape or an unordered expanded structure. Circular dichroic results were consistent with a predominantly unordered structure independently of the preparation procedure. This supports the notion that MAP2, in solution, is a very flexible and non-compact protein. Examination of the circular dichroism of MAP2 as a function of temperature indicated that the thermal stability of this protein was probably due to its mostly unordered structure and the fact that the little ordered structure present was resistant to thermal denaturation.

Vibrational circular dichroism of polypeptides, V. A study of 310‐helical‐octapeptides

Abstract: We have measured vibrational CD spectra in the 3600–1250 cm−1 region of two monodisperse, protected octapeptides, which form right-handed 310-helices in CDC13 solution. The spectra are similar in sign pattern to those obtained for right-handed α-helices in solution but are smaller in magnitude and, additionally, provide evidence of some line-shape differences. The delineation of this type of ordered conformation was accomplished by means of 1H-nmr. Such a solution structure is consistent with the x-ray crystal structure of one of these molecules.

Reversible self-association of bovine growth hormone during equilibrium unfolding.

Abstract: Previous investigations have shown that bovine growth hormone (bGH, somatotropin) unfolds through a reversible multistate process with at least one stable equilibrium intermediate. In extending our knowledge of the folding process for bGH, we demonstrate that a self-associated form of partially denatured bGH is formed during equilibrium unfolding experiments. The self-associated species has been identified by hydrodynamic measurements (size exclusion high-performance liquid chromatography and static and dynamic light scattering) and by measurements of the bGH concentration dependence of aromatic amino acid spectral properties (fluorescence, second-derivative absorption, and circular dichroism). The apparent maximum concentration for self-association occurs when bGH is partially denatured, i.e., at 3.7 M guanidine hydrochloride or 8.5 M urea, and its formation is reversible. Some of the properties of the self-associated species include quenched tryptophan fluorescence, increased tryptophan circular dichroism intensity at 300 nm, polar tryptophan environment, and a weight-average radius of about 5 nm. The self-association of bGH is mediated by specific intermolecular interactions with little increase in molecular size occurring above the saturation level of 4 mg/mL bGH. These phenomena have important implications for the design and interpretation of folding experiments in vitro and may have physiological consequences.

Characterization and structural aspects of the enhanced assembly of tubulin after removal of its carboxyl‐terminal domain

Abstract: Limited subtilisin cleavage of tubulin results in formation of S-tubulin heterodimer and a 4-kDa carboxyl-terminal peptide fragment. This carboxyl-terminal domain constitutes an essential site for MAPs interaction and plays a role in modulating the interactions responsible for tubulin self-assembly into microtubules [Serrano et al. (1984) Proc. Natl Acad. Sci. USA 81, 5989; and Biochemistry 23, 4675]. In the present communication it is shown that addition of the 4-kDa peptide fragment from porcine tubulin to porcine S-tubulin in a molar ratio of about 2:1 does not affect the assembly of the latter. On the other hand, consistent with previous findings on the binding of the 4-kDa peptide by MAP-2, the peptide inhibited MAP-2-induced tubulin assembly (molar ratio of peptide to tubulin, about 2:1; peptide to MAP-2, about 30:1). Comparison of the amino acid composition of the 4-kDa peptide fragment and the C-terminal amino acid residues of S-tubulin with the amino acid sequence of tubulin indicated the subtilisin cleavage site on the tubulin molecule to be between residues Glu417 and Phe418 of the alpha-subunit sequence and between Glu407 and Phe408 of the beta-subunit sequence. The circular dichroism of the 4-kDa fragment in water as solvent is indicative of a molecule with an unordered structure, but when the solvent is changed to a water-trifluoroethanol mixture, the fragment becomes more highly structured. The critical concentration for S-tubulin assembly is not affected by MAPs nor by polylysine, but is decreased by either taxol of dimethylsulfoxide. S-tubulin, with its greater propensity for self-association, has a different conformation from tubulin as shown by a 50% decrease in alpha-helical content, a more hydrophobic environment of at least some of the tryptophan residues as judged from fluorimetry, and a greater compaction indicated by f/f0 = 1.3, as compared to 1.4 for tubulin. The latter point is supported by the observation that the value of the sedimentation coefficient, s20,w = 5.7 S, of the 92-kDa S-tubulin molecule is not significantly different from that of the 100-kDa tubulin, s20,w = 5.8S.