Analysis of N-terminal capping using carbonyl-carbon chemical shift measurements.

The model peptide XAAAAEAAARAAAARamide is used to examine the contributions of an N-terminal capping interaction to the conformation and stability of a helical ensemble. The reference peptide has an alanine residue at position X while the capping peptide has a serine residue at this position. The helical ensemble was characterized using circular dichroism measurements and carbonyl-carbon chemical shift measurements of selectively enriched residues. The distribution of helicity within the ensemble of the reference peptide at pH 11 and 0 degrees C appears symmetrical, having a uniform central helix and frayed ends. This distribution is truncated at pH 6 by the repulsive electrostatic interaction between the positively charged alpha-amino group and the positively charged end of the helical macrodipole. The capping peptide forms a side-chain/ main-chain hydrogen bond involving the serine residue and amide of alanine 4. The presence of this hydrogen bond generates a unique motif in the chemical shift profile of its helical ensemble. The conformational stabilization contributed by this hydrogen bond, although cooperatively distributed throughout the helical ensemble, is preferentially focused within the first helical turn. The stabilization provided by this hydrogen bond is able to offset the truncation of the helical ensemble generated by the repulsive electrostatic interaction observed at pH 6.

The role of PII conformations in the calculation of peptide fractional helix content.

Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non-alpha-helical peptide acetylYEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left-handed helical conformation and unstructured denotes all the remaining conformations in a random coil ensemble. Replacement of the proline residue facilitates population of residues in an alpha-helical conformation. However, the ellipticity values for these non-proline peptides merge with the ellipticity of the proline peptide as the population of residues in the alpha-helix conformation is diminished. This convergence suggests that all residues in a host/guest peptide series of the same length share a common PII/unstructured conformational equilibrium in a given solvent. We propose that the fractional helix content of peptides within such a series may be estimated by using a two-state calculation in which the ellipticity for the non-alpha-helix conformations is provided by a peptide having a central proline guest residue.

Dichroic statistical model for prediction and analysis of peptide helicity.

Traditional statistical models for the prediction of peptide helicity are written in terms of the mean fractional helicity of the peptide residues. Far ultraviolet circular dichroic measurements of peptide solutions are converted to mean fractional helicity by partitioning the observed ellipticity between that of a perfect helix and a random coil. This partition does not adequately represent the ensemble of peptide molecules present in solution that populate imperfect helical conformations of quite variable lengths. A new dichroic statistical model has been written in terms of ellipticity rather than fractional helical content that recognizes (1) the source of ellipticity, peptide bond adsorption; (2) the differential ellipticity of peptide bonds in the terminal and interior helical turns; and (3) the contributions of each participant in a conformational ensemble to the observed ellipticity. Comparative analyses of host/guest peptides indicates that significant differences are obtained between residue w and n weights and ellipticity values using the traditional and dichroic statistical models.

Evidence for glutamate self-capping within a peptide helix.

The thermal dependence of the carbonyl carbon chemical shift of each residue in a helical peptide may be analyzed in terms of a two-state helix/coil transition. Such analyses generate values for the chemical shift of each residue in the helical and in the coil conformational ensembles of the peptide. The sequence dependence of the difference in these two values, termed the difference chemical shift, provides a description of the mean distribution of helicity within the helical ensemble. In this report, we improve two aspects of the procedures used to analyze prior chemical shift measurements of the helical peptide acetylW (EAAAR)3Aamide. The new difference chemical shift values for 16 of the 18 residues describe a very uniform central helical ensemble with frayed ends. However, the difference chemical shift values for the two remaining residues, alanines 03 and 08, are significantly diminished relative to this uniform distribution. Each of these two alanine residues is located i-4 to a glutamate residue. It is suggested that the difference chemical shifts for these two alanine residues are diminished by a self-capping interaction within the i + 4 glutamate residues.

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.

Modulation of the helical stability of a model peptide by ionic residues.

The mean residue ellipticity of the helical host peptide, acetyl-YEAAAKEAXAKEAAAKA-amide containing guest residues at position X, was measured as a function of pH and ionic strength at 0 degree C. Changes in ellipticity at 222 nm were interpreted in terms of a two-state helix/coil transition of a monomeric peptide. Variable pH measurements in low concentrations of KCl defined changes in helix stability resulting from the ionization of each guest residue. Variable [KCl] measurements at fixed pH generated ellipticity values for the neutral and ionic forms of each guest residue free of electrostatic and lyotropic contributions. These ellipticity values were used to calculate a helix propagation parameter for each form of a guest residue using the Lifson-Roig algorithm and assuming a universal nucleation parameter. In all cases, the propagation parameter of a residue is either unaffected or decreased by ionization of its side chain.

Residue helix parameters obtained from dichroic analysis of peptides of defined sequence.

Circular dichroic measurements of the host peptide acetyl-Y(EAAAK)3A-amide were obtained in solutions of increasing ionic strength at pH 7.0 and 0 degree C. The changes observed in the dichroic spectra are characteristic for a two-state helix/coil transition. The mean residue ellipticity at 222 nm exhibits a curvilinear dependence on ionic strength which becomes linear at ionic strengths greater than 1 M. The slope of the linear portion is assumed to represent the lyotropic character of the salt, and its extrapolated intercept is assumed to represent the mean residue ellipticity of the peptide solution freed from both electrostatic and lyotropic contributions which affect the helical stability of the host peptide. An extrapolated mean residue ellipticity value was obtained for each host peptide having a different amino acid guest residue at position 9 in the peptide sequence. These values were used to calculate a propagation parameter, s, for each residue using the Lifson-Roig algorithm for peptide helical content and assuming a common nucleation parameter of 0.003. The ability of these minimally determined residue parameters to predict the helical content of a variety of peptides is encouraging. Estimates were also made of the delta G values for the electrostatic interactions within the host peptide and for the additional interactions generated by ionic guest residues.

A reexamination of the conformational transitions of T4 thioredoxin.

The unfolding and refolding of T4 thioredoxin was observed by equilibrium and kinetic size exclusion chromatographic measurements in guanidine hydrochloride at 4 degrees C and pH 7.0. All the observed chromatographic profiles can be simulated by a cubic mechanism using a consistent set of equilibrium and kinetic parameters describing each of the coupled transitions. The four components in the folded protein and in the unfolded protein are interrelated by configurational transitions having parameters characteristic for proline peptide isomerizations. Only two of the four folded conformations are significantly populated at equilibrium. Each of the four unfolded components can refold by a unique conformational transition. No transiently populated folding intermediates are detected having hydrodynamic volumes intermediate between those characteristics for the folded and unfolded protein.

Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 1. Denaturant dependent baseline changes.

Size exclusion profiles of proteins with persistent conformations exhibit broad asymmetric peaks whose shape and elution times are dependent on denaturant concentration. The collective elution profiles were precisely simulated by an apparent binding model that treats the denaturant dependence in terms of an apparent matrix binding. The model requires three experimentally measurable parameters: the elution time for the unbound protein, an apparent association equilibrium constant for binding, and an apparent exchange time for binding. The denaturant dependence for each of these parameters is related to the accessible surface area of the protein.

Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 2. Comparison of spectral and chromatographic kinetic analyses.

The kinetics of the hydrodynamic volume change associated with the unfolding and refolding of a globular protein can be observed using high performance size exclusion chromatography. Chromatographic profiles that evidence such dynamics can be simulated using equations in which chromatographic partitioning and the conformational transition are described in terms of a finite difference algorithm incorporating an apparent binding model to generate broad and asymmetric peaks. Application of these equations to the simple two-state unfolding transition of ribonuclease A in guanidine hydrochloride indicates that reliable kinetic parameters can be obtained using these equations.

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.

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.

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.

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.

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.

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)

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.

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.