No abstract

Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non-a-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 a-helical conformation. However, the elliptcity values for these non-proline peptides merge with the ellipticity of the proline peptide as the population of residues in the a-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-a-helix conformations is provided by a peptide having a central proline guest residue.Keywords: ellipticity; helix content; peptide; PI1 conformation; random coil A variety of studies has examined the effect of a guest amino acid residue on the helical content of a host peptide of defined sequence (e.g., O'Neil & DeGrado, 1990;Lyu et al., 1990Lyu et al., , 1993Forood et al., 1993;Park et al., 1993; Chakrabarrty et al., 1994;Doig & Baldwin, 1995;Munoz et al., 1995). Such studies have been used to rank order the apparent helix propensity of the 20 common amino acid residues and to generate residue statistical parameters for prediction of helical content from sequence.We have utilized the sequence acetylYEAAAKEAXAKEAA AKAamide to synthesize a series of host/guest peptides, each containing a different guest residue X at sequence position 9 (Merutka et al., 1990;Park et al., 1993). These host/guest peptides generate a nested set of dichroic spectra having an isodichroic point at 203 nm and -16,500 deg cm2 dmol" at pH 7 and 0". The alanine 9 guest peptide has the most negative ellipticity at 222 nm, -24,300 deg cm2 d m o l~ I , and a dichroic spectrum characteristic for peptide solutions having a high fractional population of residues in the helical conformation. The proline 9 guest peptide has the least negative ellipticity at 222 nm, +I20 deg cmz dmol", and a dichroic spectrum characteristic for a random coil conformation.The proline 9 guest peptide was used subsequently to measure the effects of a variety of denaturing solvent conditions on the dichroic spectrum of a random coil. We were surprised to observe Reprint requests to: Earle Stellwagen, Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242; e-mail: cmdste@blue.weeg. uiowa.edu. that these solvent conditions generated a nested set of dichroic spectra characteristic for a conformational transition. We suggest that these dic...

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 two-state helix-coil model underlies all calculations of fractional helicities FH from CD spectra of helical polypeptides. The presence of an isodichroic point near 203 nm is widely assumed to validate this model, but is shown here to provide inadequate validation for alanine-rich peptides. A parametric correlation with constant slope B between CD ellipticities at a pair of wavelengths is introduced as a more rigorous two-state test. Correlations of temperature-dependent [theta](222) vs [theta](208) values are reported for a variety of peptides. Constant slopes B are observed for literature CD data obtained from fragments of helical proteins and dimeric helical coiled coils, but parametric correlations of CD data for alanine-rich peptides consistently exhibit anomalous concave upward curvature, characterized by local slopes that are linearly temperature dependent. Low-temperature CD studies together with parametric correlations at a series of wavelengths demonstrate that the curvature anomaly is maximal at 222 nm and localized in the 215-230 nm wavelength region. Precedented structural variation of the phi, psi dihedral angles of the alpha-helix is suggested as a possible explanation. For the important case of alanine-rich peptides, experiments are proposed that may yield temperature corrections for [theta](222) and permit reliable calculations of FH from [theta](222) values.

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.