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E Stellwagen

Publications and source records attributed to E Stellwagen.

At least 19 recordsLinked to original sources
PubMed1995

Incorporation of pairwise interactions into the Lifson-Roig model for helix prediction.

The helix/coil equilibrium of a peptide in solution can be modulated by a variety of side-chain interactions that are not incorporated into the standard statistical mechanical models for prediction of peptide helical content. In this report, we describe a recursive formulation of the Lifson-Roig model that facilitates incorporation of specific pairwise side-chain interactions as well as nonspecific individual side-chain capping interactions. Application of this extended model to a series of host/guest peptides indicates that the apparent delta G value for a pairwise apolar interaction is dependent upon the spacing and orientation but not the sequential location of the participating residues. The apparent delta G values for such interactions are about 40% greater than the corresponding apparent delta delta G values obtained from difference measurements.

Amino Acid Sequence↗
PubMed1992

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.

Chromatography, Gel↗
PubMed1992

The contribution of residue ion pairs to the helical stability of a model peptide.

Comparative CD measurements were made on the model helical peptides acetylYEAAAKEAXAKEAAAKAamide and acetylYEAAAEKAXAKEAAAKAamide in which X represents a nonaromatic nonionic residue. The former peptide contains three potential i, i + 4 complementary ion pairs at neutral pH, while the latter peptide contains one potential complementary and two potential antagonistic i, i + 4 ion pairs. The effect of pH and ionic strength on the mean residue ellipticity of these peptides was measured at 222 nm and 0 degrees C. These measurements were analyzed assuming a common two-state helix/coil transition and only i, i, + 4 ion-pair interactions. The analyses suggest that the central ion pairs do modulate helical content while the peripheral ion pairs do not, presumably due to the location of the peripheral ion pairs in the frayed ends of the helix. The complementary central ion pair stabilizes the helix by about 0.4 kcal/mole and the antagonistic central ion pair destabilizes the helix by about 0.2 kcal/mole.

Amino Acid Sequence↗
PubMed1991

A model peptide with enhanced helicity.

The sequence of a model monomeric peptide, acetylA(EAAAK)3Aamide was altered to expedite measurement of peptide concentration and to enhance its fractional helical content. Replacement of the N-terminal alanine residue with a tryptophan residue provides a convenient chromophore for measurement of peptide concentration without diminishing the helical content. Replacement of the three lysine residues with arginine residues enhances the helical content without loss of their electrostatic contributions. Increasing the number of EAAAR sequence units in the peptide acetylW(EAAAR)nAamide from three to five indicates that the spectral features anticipated for a completely helical peptide are closely approached.

Alanine↗
PubMed1991

Effect of amino acid ion pairs on peptide helicity.

The three ER ion pairs in the peptide acetyl-W(EAAAR)3A-amide were replaced in turn with the ion pairs EK, EO, DR, DK, and DO, where O represents an ornithine residue. The far-ultraviolet circular dichroic spectra of the six peptides measured in 10 mM NaCl at pH 2 and 0 degrees C form a nested set having an isodichroic point at 203 nm of -17,000 deg cm2 dmol-1. The ellipticity values of the six peptides at 222 nm range from -31,600 to -7400 deg cm2 dmol-1 in the order listed. Changing the pH of each peptide solution from 2 to 13 also generates a nested set of dichroic spectra with the same isodichroic values. Increasing the pH from 2 to 7 differentially increases the ellipticity at 222 nm in a single transition having an apparent pK of 4.1 for the E-containing peptides are 3.6 for the D-containing peptides. Increasing the pH beyond neutrality differentially decreases the ellipticity at 222 nm in a single transition having an apparent pK of greater than or equal to 13.2 for the R-containing peptides, 11.1 for the K-containing peptides, and 10.7 for the O-containing peptides. It is proposed that the difference in the ellipticity of the six peptides chiefly reflects the helix preferences for the variable residues supplemented by intrahelical electrostatic interactions in the neutral pH range.

Amino Acid Sequence↗
PubMed1990

Effect of central-residue replacements on the helical stability of a monomeric peptide.

The peptide acetylYEAAAKEARAKEAAAKAamide exhibits the dichroic features characteristic of a monomeric helix/coil transition in aqueous solution. Nineteen variants of this peptide each containing a different residue at position 9 were prepared by solid-phase peptide synthesis and purified by reversed-phase chromatography. The thermal dependence of the far-ultraviolet dichroic spectrum of each of these peptides except that containing proline is characteristic for an alpha-helix/coil transition. The relative stability of the helical forms of these peptides does not correlate with the preference of the variable amino acid to occupy a middle position in a protein helix. It is likely that the specific interactions of the variable residue with its local environment obscure any inherent preference of the residue to reside in an alpha-helix.

Amino Acid Sequence↗
PubMed1990

Positional independence and additivity of amino acid replacements on helix stability in monomeric peptides.

The 17-residue peptide acetylAEAAAKEAAAKEAAAKAamide, described as an autonomous folding unit (Marqusee & Baldwin, 1987), has been used to examine the effect of amino acid replacements on helix stability. Alanine residues(s) at positions 4, 9, and 14 in the peptide sequence were replaced either singly or multiply by either serine or methionine residues with solid-phase peptide synthesis. The thermal dependence of the helix/coil transition of each peptide was observed by far-ultraviolet circular dichroism. Within experimental variation, all three single replacements exhibit a common thermal transition, and all three double replacements exhibit a different common thermal transition. These results suggest that replacement of the central alanine residue in the repeat EAAAK located in the N-terminus, in the middle, or in the C-terminus of the peptide helix has the same effect on helix stability. The melting temperature of each thermal transition was estimated by assuming a linear van't Hoff plot and a change in molar ellipticity of 33,500 deg cm2 dmol-1. Such analysis indicates that each replacement of an alanine residue by a serine residue diminishes the melting temperature by 11 +/- 1 degrees C and that each replacement of an alanine residue by a methionine residue diminishes the melting temperature by 6 +/- 1 degrees C. These results suggest that the effect of these replacements on helix stability is additive.

Alanine↗
PubMed1990

Gel filtration.

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Chromatography, Gel↗
PubMed1989

Refolding of denatured ribonuclease observed by size exclusion chromatography.

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.

Animals↗
PubMed1989

Analysis of peptides for helical prediction.

Two terminally blocked peptides, acetylAETAAAKFLRQHMamide and acetylAETSSSRYLRQHMamide, were obtained by solid-phase synthesis, purified by reversed-phase chromatography, and characterized by fast atom bombardment mass spectrometry. Both peptides were soluble in aqueous solutions and remained monomeric over the concentration range examined. Changes in the temperature, pH, and trifluoroethanol concentration of solutions of each peptide produced changes in the far-ultraviolet circular dichroic spectrum characteristic of a two-state helix/coil transition. The limiting mean residue ellipticity of the coil and helix form of each peptide was estimated by addition of the denaturant guanidinium chloride at elevated temperature and by addition of trifluoroethanol at subzero temperatures, respectively. The midpoint for the thermal transition of the peptide SSSRY is lowered by about 30 degrees C relative to that of peptide AAAKF, in qualitative agreement from predictions based on helix probabilities of amino acid residues. The magnitude of the change observed in the midpoint of the thermal transitions suggests that the effect of single amino acid replacements on helix formation should be experimentally measurable.

Amino Acid Sequence↗
PubMed1987

Refolding of denatured thioredoxin observed by size-exclusion chromatography.

Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1987

Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.

The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.

Bacterial Proteins↗
PubMed1987

Accessibility and multivalency of immobilized Cibacron blue F3GA.

The effect of immobilized dye concentration on protein complexation was observed using zonal chromatography. A monomeric protein, octopine dehydrogenase, was retained by a single interaction to a Sepharose CL-6B column containing 11.6 mM immobilized Cibacron blue F3GA. By contrast, a tetrameric protein, lactate dehydrogenase, was retained by the same column by multiple interactions. The degree of multiple interactions was found to systematically increase with increasing immobilized dye concentration. The concentration of immobilized dye accessible to protein was found to be inversely related to the concentration of ionic components in the solvent. Zonal chromatographic measurements of free dye and unconjugated matrix suggest that increasing the concentration of ionic components promotes the adsorption of immobilized dye to the adjacent matrix surface. Such adsorption markedly affects both the capacity of an immobilized dye column and the multiplicity of its interaction with oligomeric proteins.

Enzymes, Immobilized↗
PubMed1986

Equilibrium and kinetic measurements of the conformational transition of thioredoxin in urea.

Addition of urea to solutions of Escherichia coli thioredoxin results in a cooperative unfolding of the protein centered at 6.7 M urea at 25 degrees C and 5.1 M urea at 2 degrees C and neutral pH as judged by changes in tryptophan fluorescence emission, far-ultraviolet circular dichroism, and exclusion chromatography. Kinetic profiles of changes in tryptophan fluorescence emission intensity were analyzed following either manual or stopped-flow mixing to initiate unfolding or refolding. Unfolding of the native protein occurs in a single kinetic phase whose time constant is markedly dependent on urea concentration. Refolding of the urea-denatured protein occurs in a multiplicity of kinetic phases whose time constants and fractional amplitudes are also dependent upon urea concentration. Urea gradient gel electrophoretic and exclusion chromatographic measurements suggest the transient accumulation of at least one and likely two compact nativelike intermediate conformations during refolding. Simulations of both electrophoretic and chromatographic results suggest that the intermediate conformations are generated by the concerted action of the middle and fast refolding phases.

Bacterial Proteins↗
PubMed1986

Zonal chromatographic analysis of the interaction of alcohol dehydrogenase with blue-sepharose.

The interaction between horse liver alcohol dehydrogenase and Reactive blue 2 immobilized on Sepharose CL-6B was measured by zonal chromatography. Each protein molecule was retained by a single immobilized dye using a Blue-Sepharose column containing a total of 1.38 mM dye. However, the protein was predominantly retained by two immobilized dye molecules using a darker Blue-Sepharose column containing a total of 11.6 mM dye. The dissociation constant measured for the alcohol dehydrogenase--immobilized dye complex on each column is identical to the inhibition constant for the alcohol dehydrogenase--free Reactive blue 2 complex: 4.5 +/- 0.8 microM.

Alcohol Oxidoreductases↗
PubMed1986

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin.

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.

Bacterial Proteins↗
PubMed1985

Refolding a disulfide dimer of cytochrome c.

A covalent dimer of Saccharomyces cerevisiae iso-1 cytochrome c is stabilized by an interchain disulfide bond involving the cysteine residue penultimate to the C-terminus. The individual chains in the dimer appear to retain the tertiary structural features characteristic for monomeric cytochrome c albeit with some perturbation. The dimer is reversibly denatured by heat, urea, or guanidine hydrochloride in a single cooperative transition whose midpoint is less than that of the monomeric protein. The kinetic profile observed for the refolding of the denatured dimer is characteristic for monomeric cytochromes except for a markedly enhanced slow-phase amplitude.

Circular Dichroism↗