Studies of the minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes.

The effects of the hydrophobicity and the distribution of hydrophobic residues on the surfaces of some designed alpha-helical transmembrane peptides (acetyl-K2-L(m)-A(n)-K2-amide, where m + n = 24) on their solution behavior and interactions with phospholipids were examined. We find that although these peptides exhibit strong alpha-helix forming propensities in water, membrane-mimetic media, and lipid model membranes, the stability of the helices decreases as the Leu content decreases. Also, their binding to reversed phase high-performance liquid chromatography columns is largely determined by their hydrophobicity and generally decreases with decreases in the Leu/Ala ratio. However, the retention of these peptides by such columns is also affected by the distribution of hydrophobic residues on their helical surfaces, being further enhanced when peptide helical hydrophobic moments are increased by clustering hydrophobic residues on one side of the helix. This clustering of hydrophobic residues also increases peptide propensity for self-aggregation in aqueous media and enhances partitioning of the peptide into lipid bilayer membranes. We also find that the peptides LA3LA2 [acetyl-K2-(LAAALAA)3LAA-K2-amide] and particularly LA6 [acetyl-K2-(LAAAAAA)3LAA-K2-amide] associate less strongly with and perturb the thermotropic phase behavior of phosphatidylcholine bilayers much less than peptides with higher L/A ratios. These results are consistent with free energies calculated for the partitioning of these peptides between water and phospholipid bilayers, which suggest that LA3LA2 has an equal tendency to partition into water and into the hydrophobic core of phospholipid model membranes, whereas LA6 should strongly prefer the aqueous phase. We conclude that for alpha-helical peptides of this type, Leu/Ala ratios of greater than 7/17 are required for stable transmembrane associations with phospholipid bilayers.

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour.

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.

The perchlorate anion is more effective than the trifluoroacetate anion as an ion-pairing reagent for reversed-phase chromatography of peptides.

The addition of salts, specifically sodium perchlorate (NaClO4), to mobile phases at acidic pH as ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) has been generally overlooked. To demonstrate the potential of NaClO4 as an effective anionic ion-pairing reagent, we applied RP-HPLC in the presence of 0-100 mM sodium chloride (NaCl), sodium trifluoroacetate (NaTFA) or NaClO4 to two mixtures of synthetic 18-residue peptides: a mixture of peptides with the same net positive charge (+4) and a mixture of four peptides of +1, +2, +3 and +4 net charge. Interestingly, the effect of increasing NaClO4 concentration on increasing peptide retention times and selectivity changes was more dramatic than that of either NaCl or NaTFA, with the order of increasing anion effectiveness being Cl- << TFA- < C104-. Such effects were more marked when salt addition was applied to eluents containing 10 mM phosphoric acid (H3PO4) compared to 10 mM trifluoroacetic acid (TFA) due to the lesser starting anion hydrophobicity of the former mobile phase (containing the phosphate ion) compared to the latter (containing the TFA- ion).

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography.

Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.

Effect of anionic ion-pairing reagent concentration (1-60 mM) on reversed-phase liquid chromatography elution behaviour of peptides.

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).

Stability and specificity of heterodimer formation for the coiled-coil neck regions of the motor proteins Kif3A and Kif3B: the role of unstructured oppositely charged regions.

We investigated the folding, stability, and specificity of dimerization of the neck regions of the kinesin-like proteins Kif3A (residues 356-416) and Kif3B (residues 351-411). We showed that the complementary charged regions found in the hinge regions (which directly follow the neck regions) of these proteins do not adopt any secondary structure in solution. We then explored the ability of the complementary charged regions to specify heterodimer formation for the neck region coiled-coils found in Kif3A and Kif3B. Redox experiments demonstrated that oppositely charged regions specified the formation of a heterodimeric coiled-coil. Denaturation studies with urea demonstrated that the negatively charged region of Kif3A dramatically destabilized its neck coiled-coil (urea1/2 value of 3.9 m compared with 6.7 m for the coiled-coil alone). By comparison, the placement of a positively charged region C-terminal to the neck coiled-coil of Kif3B had little effect on stability (urea1/2 value of 8.2 m compared with 8.8 m for the coiled-coil alone). The pairing of complementary charged regions leads to specific heterodimer formation where the stability of the heterodimeric neck coiled-coil with charged regions had similar stability (urea1/2 value of 7.8 m) to the most stable homodimer (Kif3B) with charged regions (urea1/2 value of 8.0 m) and dramatically more stable than the Kif3A homodimer with charged regions (urea1/2, value of 3.9 m). The heterodimeric coiled-coil with charged extensions has essentially the same stability as the heterodimeric coiled-coil on its own (urea1/2 values of 7.8 and 8.1 m, respectively) suggesting that specificity of heterodimerization is driven by non-specific attraction of the oppositely unstructured charged regions without affecting stability of the heterodimeric coiled-coil.

Monitoring the hydrophilicity/hydrophobicity of amino acid side-chains in the non-polar and polar faces of amphipathic alpha-helices by reversed-phase and hydrophilic interaction/cation-exchange chromatography.

The ability to monitor precisely the hydrophobicity/hydrophilicity effects of amino acid substitutions in both the non-polar and polar faces of amphipathic alpha-helical peptides is critical in such areas as the rational de novo design of more effective antimicrobial peptides. The present study reports our initial results of employing the complementary separation modes of reversed-phase high-performance liquid chromatography (RP-HPLC) and hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) to monitor the effect on apparent peptide hydrophilicity/hydrophobicity and amphipathicity of substituting single L- or D-amino acids into the centre of the non-polar or polar faces of a 26-residue biologically active amphipathic alpha-helical peptide, V681. Our results clearly show that RP-HPLC and HILIC/CEX are best suited for resolving amphipathic peptides where substitutions are made in the non-polar and polar faces, respectively. Further, RP-HPLC and HILIC/CEX were demonstrated to be excellent monitors of hydrophilicity/hydrophobicity variations where amino acid substitutions were made in these respective faces. We believe these complementary high-performance modes offer excellent potential for rational design of novel amphipathic alpha-helical biologically active peptides.

Optimum concentration of trifluoroacetic acid for reversed-phase liquid chromatography of peptides revisited.

Trifluoroacetic acid (TFA) remains the dominant mobile phase additive for reversed-phase high-performance liquid chromatography (RP-HPLC) of peptides after more than two decades since its introduction to this field. Generally, TFA has been employed in a concentration range of 0.05-0.1% (6.5-13 mM) for the majority of peptide separations. In order to revisit the question as to whether such a concentration range is optimum for separations of peptide mixtures containing peptides of varying net positive charge, the present study examined the effect of varying TFA concentration on RP-HPLC at 25 and 70 degrees C of three groups of synthetic 10-residue synthetic peptides containing either one (+1) or multiple (+3, +5) positively charged groups. The results show that the traditional range of TFA concentrations employed for peptide studies is not optimum for many, perhaps the majority, of peptide applications. For efficient resolution of peptide mixtures, particularly those containing peptides with multiple positive charges, our results show that 0.2-0.25% TFA in the mobile phase will achieve optimum resolution. In addition, the use of high temperature as a complement to such TFA concentration levels is also effective in maximizing peptide resolution.

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature.

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.

Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix.

We describe here a systematic study to determine the effect on secondary structure of d-amino acid substitutions in the nonpolar face of an amphipathic alpha-helical peptide. The helix-destabilizing ability of 19 d-amino acid residues in an amphipathic alpha-helical model peptide was evaluated by reversed-phase HPLC and CD spectroscopy. l-Amino acid and d-amino acid residues show a wide range of helix-destabilizing effects relative to Gly, as evidenced in melting temperatures (DeltaTm) ranging from -8.5 degrees C to 30.5 degrees C for the l-amino acids and -9.5 degrees C to 9.0 degrees C for the d-amino acids. Helix stereochemistry stability coefficients defined as the difference in Tm values for the l- and d-amino acid substitutions [(DeltaTm' = TmL and TmD)] ranging from 1 degrees C to 34.5 degrees C. HPLC retention times [DeltatR(XL-XD)] also had values ranging from -0.52 to 7.31 min at pH 7.0. The helix-destabilizing ability of a specific d-amino acid is highly dependent on its side-chain, with no clear relationship to the helical propensity of its corresponding l-enantiomers. In both CD and reversed-phase HPLC studies, d-amino acids with beta-branched side-chains destabilize alpha-helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure-activity studies and de novo design of novel biologically active peptides.

Development of simultaneous purification methodology for multiple synthetic peptides by reversed-phase sample displacement chromatography.

We have developed a low-pressure protocol, designed as a rapid, simple and cost-effective procedure for the efficient and parallel purification of multiple peptide mixtures. This was achieved through adaptation of our novel reversed-phase sample displacement chromatography (SDC) method, where the major separation process takes place in the absence of organic modifier, to modular solid-phase extraction (SPE) technology. Thus, crude peptide sample is applied at overload conditions to extraction columns consisting of SPE tubes containing silica-based reversed-phase packing. By applying a vacuum to draw the solution through the packing, product separation from hydrophobic and hydrophilic impurities is accomplished in a two-stage purification unit: a short pre-column functions as a trap for hydrophobic impurities, while a second, longer SPE column is used as a product isolation column. Thus, under ideal SDC conditions, washing with a 100% aqueous solvent will achieve retention of hydrophobic impurities on the trap, with displacement of product and hydrophilic impurities from the trap to the product isolation column; hydrophilic impurities are thus displaced off the product isolation to waste, leaving only product retained on the main column. In this initial evaluation, this purification system has demonstrated excellent separation of product, in good yield, from both hydrophilic and hydrophobic impurities over a wide range of peptide hydrophobicity and crude composition for model synthetic peptide systems representing crude peptide mixtures.

Trapping the monomeric alpha-helical state during unfolding of coiled-coils by reversed-phase liquid chromatography.

Reversed-phase liquid chromatography (RPLC) offers a unique opportunity to monitor the transition from the native state (N) to the structural intermediate state (I) for proteins whose secondary structure is comprised entirely of amphipathic helices, such as coiled-coils. During RPLC, the hydrophobicity of the stationary phase and mobile phase results in the unfolding of the tertiary/quaternary structure of coiled-coils but retains alpha-helical secondary structure and thus isolates the I state. A set of five peptides, alphaalpha-36, betabeta-36, alphabeta-36, gammadelta-36 and omegaomega-36, was generated by shuffling guest hydrophobes at equivalent sites in a symmetric host frame. In one of the peptides, omegaomega-36, all the alpha-glutamic residues in the host frame were replaced by gamma-glutamic residues. alphaalpha-36, betabeta-36, alphabeta-36, gammadelta-36 form two-stranded coiled-coils of identical helical content and unfold as a two-state transition during temperature denaturation while the fifth peptide, omegaomega-36, is a random coil and cannot be induced in to an alpha-helical structure even in the presence of a helix inducing solvent, 50% trifluoroethanol. By comparing the stability order of the four coiled-coils in the N-->I transition (measured by RPLC studies) with that in the N-->D (denatured state) transition (measured by calorimetry), it is concluded that there is a direct correlation between the relative stabilities of these peptides in these two unfolding transitions. This result supports a hierarchical folding mechanism for coiled-coils.

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d".

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.

Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side-chain acetylated peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation-exchange matrix. Complex peptide mixtures produced by solid-phase synthesis are a frequently encountered and challenging purification problem. In the present study a two-step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21-residue synthetic amphipathic alpha-helical peptide from serine side-chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid-phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the alpha-helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.

Hydrophilic interaction/cation-exchange chromatography for separation of amphipathic alpha-helical peptides.

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which has excellent potential for peptide separations. Separations by HILIC/CEX are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlaid on ionic interactions with the cation-exchange matrix. In the present study, HILIC/CEX has been applied to the separation of synthetic amphipathic alpha-helical peptides, varying in amphipathicity and the nature of side-chain substitutions in the centre of the hydrophobic or hydrophilic face. Observation of the retention behaviour of these amphipathic alpha-helical peptide analogues during HILIC/CEX and reversed-phase chromatography (RPLC) enabled the establishment of general rules concerning the applicability of these complementary HPLC techniques to peptides displaying a secondary structural motif of common occurrence.

Structural cassette mutagenesis in a de novo designed protein: proof of a novel concept for examining protein folding and stability.

The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.

Use of sodium perchlorate at low pH for peptide separations by reversed-phase liquid chromatography. Influence of perchlorate ion on apparent hydrophilicity of positively charged amino acid side-chains.

The reversed-phase liquid chromatography (RPLC) behavior of synthetic model peptides containing positively charged amino acid residues was studied in the presence or absence of 100 mM sodium perchlorate in order to determine the effect on apparent side-chain hydrophilicity of a charged residue at low pH. The peptides used in this study were either non-helical peptides or amphipathic alpha-helical peptides, where the effect of the negatively charged perchlorate ion on a charged residue in either the hydrophobic face or hydrophilic face of the helix was monitored. We have shown that the addition of 100 mM perchlorate to RPLC separations of positively charged peptides performed in a 20 mM aqueous phosphoric acid-acetonitrile system resulted in an increase in retention time of a peptide relative to the same peptide in the absence of perchlorate. This effect occurred independent of conformation, i.e., whether comparing the effect of positively charged residue substitutions in the hydrophobic or hydrophilic face of an amphipathic alpha-helix or in a peptide with negligible secondary structure. From these results, suggesting that positively charged side-chain hydrophilicity is decreased by ion-pairing with the perchlorate ion, we have shown practical examples where mixtures of non-helical and amphipathic alpha-helical peptides showed enhanced resolution in the presence of perchlorate at pH 2, compared to in its absence. In addition, it was shown that an aqueous phosphoric acid-perchlorate-acetonitrile mobile phase may show markedly different selectivity for peptide separations at low pH compared to the more traditional aqueous trifluoroacetic acid-acetonitrile system.

Effect of mobile phase on the oligomerization state of alpha-helical coiled-coil peptides during high-performance size-exclusion chromatography.

Important structural motifs involving amphipathic helices include two-stranded and multiple-stranded coiled-coils. High-performance size-exclusion chromatography (HPSEC) is a useful tool to examine both the oligomerization state of coiled-coils as well as the stability of such motifs, due to the facile manipulation of the mobile phase and the lack of interaction of the peptide solutes with the stationary phase. In the present study, HPSEC was applied to two series of de novo designed model amphipathic alpha-helical peptides with the sequences (1) Ac-(E-A-L-K-A-E-I)n-E-A-C-K-A-amide, where n = 1 or 3, Ac-E-I-(E-A-L-K-A-E-I)4-E-A-C-K-A-amide and (2) Ac-(K-L-E-A-L-E-A)n-amide, where n = 1, 2 or 4. Observation of the retention behaviour of Series 1 under both denaturing and non-denaturing conditions at pH 7.0 offered insights into the effect of polypeptide chain length and disulphide bridge formation on the stability of alpha-helical coiled-coils. In contrast, the Series 2 peptides showed promise as peptide standards to monitor the effect of environment on the multi-strandedness of coiled-coils, since the 28-residue peptide of this series was eluted as a monomer, dimer or trimer depending on mobile phase conditions.