Biomedical subjects

N E Zhou

Publications and source records attributed to N E Zhou.

At least 19 recordsLinked to original sources

PubMed1996

Formation of parallel and antiparallel coiled-coils controlled by the relative positions of alanine residues in the hydrophobic core.

The orientation of alpha-helical chains in two-stranded coiled-coils has been shown to be determined by the presence of favorable interchain electrostatic interactions. In this study, we used de novo designed 35-residue peptides to show that when interchain electrostatic interactions are not a factor in coiled-coil formation, the relative positions of Ala residues in the middle heptad can control the parallel or antiparallel orientation of alpha-helical chains in coiled-coils. The peptides formed four-stranded coiled-coils where the helices are either all-parallel or all-antiparallel with respect to their nearest neighbor. The common structural element in these four-stranded coiled-coils is an alternating pair of Ala and Leu residues (Ala-Leu-Ala-Leu) in each of the two planes in the middle heptad. These results indicate that both the relative positions of the Ala residues in the hydrophobic core and the interchain electrostatic interactions between charged residues in the e and g positions should be considered in designing coiled-coils with the desired number of strands in the multiple-stranded assembly. These design elements are also important in orienting functional groups or domains attached to the terminals ends of a coiled-coil carrier.

Alanine↗

PubMed1994

Reversed-phase liquid chromatography as a useful probe of hydrophobic interactions involved in protein folding and protein stability.

We have evaluated the potential of reversed-phase liquid chromatography (RPLC) as a probe of hydrophobic interactions involved in protein folding and stability. Our approach was to apply RPLC to a de novo designed model protein system, namely a two-stranded alpha-helical coiled coil. It was shown that the reversed-phase retention behaviour of various synthetic analogues of monomeric alpha-helices and dimeric coiled-coil structures correlated well with their stability in solution, as monitored by circular dichroism during guanidine hydrochloride and temperature denaturation studies. In addition, an explanation is offered as to why amphipathic coiled coils, an important structural motif in many biological systems, are more stable at low pH compared to physiological pH values. The results of this study suggest that not only may RPLC prove to be a useful and rapid complementary technique for understanding protein interactions, but also the de novo designed coiled-coil model described here is an excellent model system for such studies.

Amino Acid Sequence↗

PubMed1994

The role of interhelical ionic interactions in controlling protein folding and stability. De novo designed synthetic two-stranded alpha-helical coiled-coils.

The role of interchain ionic interactions in controlling protein folding and stability has been studied by using de novo designed synthetic two-stranded alpha-helical coiled-coils. The model coiled-coil (denoted as EK) consists of two identical 35-residue polypeptide chains with a heptad repeat KgLaGbAcLdEeKf and a Cys residue at position 2 and an Ala residue at position 16 in each chain. The Lys residues at positions "g" in one chain and Glu residues at positions "e" in the other chain are expected to form interchain ion-pairs in the coiled-coil structure. This peptide forms a stable coiled-coil structure in benign medium (50 mM KCl, 25 mM PO4, pH7) with a [urea]1/2 value of 3.5 M. In contrast, two peptide analogs EE (EgLaGbAcLdEeKf) and KK (KgLaGbAcLdKeEf), which differ from EK in that EE contains only negatively charged Glu residues and KK contains only positively charged Lys residues at both positions e and g, each show a random coil structure in benign buffer. However, peptide EE or KK can form a stable coiled-coil structure if the interchain ionic repulsions are effectively suppressed either by changing pH or by using high salt concentrations. An equimolar mixture of these two peptides displays 100% alpha-helical content under the same conditions. These results demonstrate that although the interhelical ionic attractions are not essential for coiled-coil formation, a large number of these weak interactions can play an important role in the assembly of helices. Though interhelical ionic repulsions destabilize the homo-stranded coiled-coil, electrostatic attractions may stabilize the hetero-stranded coiled-coil. In addition, this study also suggests that the folding process for these synthetic model coiled-coils does not involve a single-stranded alpha-helix as a significantly populated folding intermediate.

Amino Acid Sequence↗

PubMed1994

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability.

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability has been quantitated using de novo designed synthetic coiled-coils. The synthesized model coiled-coil (EK), denoted by amino acid residues in positions e and g, which contains only interhelical ionic interactions without any possible (i, i + 3) and (i, i + 4) intrahelical ionic interaction, consists of two identical 35 residue polypeptide chains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coils were prepared where five Glu residues at e positions in EK were mutated to Gln residues (QK); five Lys residues at g positions were altered to Gln residues (EQ) or these mutations were effected at both positions e and g (QQ). The stabilities of the four coiled-coils were determined by measuring the ellipticities at 220 nm as a function of urea concentration at 20 degrees C. By using a double-mutant cycle analysis it was possible to isolate the energetic contribution of interhelical ionic attractions to coiled-coil stability from the other contributions such as helical preference and hydrophobicity. The 0.37 +/- 0.01 kcal/mol of energetic contribution of one interhelical ion pair to the coiled-coil stability was obtained from three independent comparisons. This findings suggests that a large number of weak interhelical electrostatic interactions on the surface of a protein can make a substantial contribution to protein stability. In addition, the energetic contributions of a single mutation E(-)-->Q, K(+)-->Q, Q-->E degrees and E(-)-->E degrees were also determined (delta delta G = 0.22, 0.26, and 0.46 and 0.65 kcal/mol for the single mutations, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Comparison of antiparallel and parallel two-stranded alpha-helical coiled-coils. Design, synthesis, and characterization.

An antiparallel coiled-coil has been designed and characterized as a model for studying protein folding and assembly. This heterostranded antiparallel coiled-coil was formed by an interchain disulfide bond between cysteine residues at position 2 of one chain and at position 33 of the other chain. Each peptide chain has 35 residues which are composed of five heptad repeats of the sequence K-L-E-A-L-E-G with a single Leu-->Ala substitution at position 16. Two homostranded parallel coiled-coils were also formed as co-products of the oxidation reaction to form the interchain disulfide bond. The CD spectra of the parallel and antiparallel peptides were very similar and their high molar ellipticities at 220 nm did not increase in the presence of 50% trifluoroethanol. These data suggest that, like the parallel peptides, the antiparallel peptide also exists in a coiled-coil structure. Urea and guanidine hydrochloride denaturation studies, in conjunction with molecular modeling studies, suggest that there are no physical restrictions to the packing of hydrophobic residues in an antiparallel coiled-coil. However, interchain electrostatic interactions can have positive or negative contributions to the overall stability of the disulfide-bridged coiled-coil. In addition, interchain electrostatic interactions appear to play a major role in protein folding by controlling the parallel or antiparallel alignment of the alpha-helical polypeptide chains. This study is also for the first time providing us with a new understanding of the information that can be obtained from urea and guanidine hydrochloride denaturation studies of proteins concerning the contributions of hydrophobic and electrostatic interactions on stability.

Amino Acid Sequence↗

PubMed1993

A single-stranded amphipathic alpha-helix in aqueous solution: design, structural characterization, and its application for determining alpha-helical propensities of amino acids.

In order to investigate the positional effect of alpha-helical propensities of amino acids in an amphipathic alpha-helix, an amphipathic alpha-helical model peptide (Ac-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys- amide) was designed and characterized by circular dichroism and 2D-NMR spectroscopies. This peptide contains 65% alpha-helical structure in solution, and its monomeric molecular weight in aqueous solution was determined by size-exclusion chromatography. The independence of alpha-helical structure and stability on peptide concentration demonstrates that helix formation of this peptide is a monomolecular process. To compare the effect of substitutions in the hydrophobic and hydrophilic face of the helix on structure and stability, a leucine, alanine, or glycine was individually substituted in the hydrophobic face (position 9) or hydrophilic face (position 7) of the model peptide. The change in helix content and stability upon substitution was measured by circular dichroism studies in the absence and presence of TFE or urea. The results indicate that each amino acid has a different helix propensity when it is located in the hydrophobic face versus hydrophilic face and the effect of substitution is more significant in the hydrophobic face. This single-stranded amphipathic alpha-helical peptide provides an appropriate model system to determine helix propensities of amino acids on both hydrophobic and hydrophilic faces.

Amino Acid Sequence↗

PubMed1993

Disulfide bond contribution to protein stability: positional effects of substitution in the hydrophobic core of the two-stranded alpha-helical coiled-coil.

To investigate the positional effect of the disulfide bond on the structure and stability of a two-stranded alpha-helical coiled-coil, an interchain disulfide bond was systematically introduced into the hydrophobic core of a de novo designed model coiled-coil at the N-terminus (position 2), C-terminus (position 33), and nonterminal positions a (positions 9, 16, 23, and 30) and d (positions 5, 12, 19, and 26). The rate of formation of a disulfide bond is faster at position d compared to at the corresponding position a under nondenaturing conditions, suggesting that position d is more suitable for engineering a disulfide bond. The structure and stability of the reduced and oxidized coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. Our results demonstrate that the improvement of protein stability by introduction of a disulfide bond is very relevant to its location and the most effective disulfide bonds are those that can be introduced in the hydrophobic core without any disruption of the protein structure. The disulfide bond at position d with near-optimal geometry does not perturb the coiled-coil structure and makes the largest contribution to coiled-coil stability. In contrast, the inappropriate geometry of the disulfide bond at nonterminal position a introduces a high strain energy on the disulfide bond which disrupts the coiled-coil structure. At positions a, the closer the disulfide bridge is to the center of the coiled-coil, the larger the disruption on the coiled-coil structure and the smaller the contribution the disulfide bond makes to coiled-coil stability. The computer modeling results also suggest that an insertion of an interchain disulfide bond at position a in the GCN4 leucine zipper X-ray structure has a higher potential energy than insertion at position d. The energy-minimized coiled-coil structure with an interchain disulfide bond at position a has a larger root mean square difference from the X-ray structure of GCN4 than the coiled-coil with a disulfide bond at position d. Because interhelical interactions are common in globular proteins as well as coiled-coils, the results obtained in this study will have general utility for selecting the sites for engineering disulfide bonds between alpha-helices.

Amino Acid Sequence↗

PubMed1993

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."

Amino Acid Sequence↗

PubMed1993

Structure, function and application of the coiled-coil protein folding motif.

Recent X-ray analyses and synthetic model studies of the coiled-coil motif have clarified roles for hydrophobic core residues and ionic interactions in determining stability, selectivity, stoichiometry and orientation of alpha-helices in this structure. Although much remains to be learnt, current knowledge now enables this motif to be used in novel constructs and points the way to a more explicit understanding of native coiled-coil formation and protein folding in general.

Amino Acid Sequence↗

PubMed1992

Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil.

Our de novo designed coiled-coil model protein consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment via interchain hydrophobic interactions and a disulfide bridge at the position 2 between two helices. To quantitate the relative contribution of leucine residues at the nonequivalent position of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil, a single alanine was systematically substituted for a leucine in each chain at position "a" (9, 16, 23, or 30) or "d" (5, 12, 19, 26, or 33). The formation and stability of the coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. All the proteins with an alanine substituted at position a have a similar stability ([Gdn.HCl]1/2 ranges from 2.6 to 2.9 M), while all the proteins with an alanine substituted at position d have similar stability ([Gdn.HCl]1/2 ranges from 3.6 to 4.2 M), except for the proteins with an alanine substituted in the C-terminal heptad. The greater decrease in stability observed for a Leu----Ala mutation at position a (the average delta delta Gu value is 3.3 kcal/mol) compared to those where the substitution was effected at position d (the average delta delta Gu value is 2.0 kcal/mol) indicates that an Ala mutation at position a has a greater effect on the side-chain packing and hydrophobic interactions in the coiled-coil than an Ala mutation at position d.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Synthetic model proteins. Positional effects of interchain hydrophobic interactions on stability of two-stranded alpha-helical coiled-coils.

We have designed a model protein that consists of two identical 35-residue polypeptide chains, parallel and in-register arranged in a two-stranded alpha-helical coiled-coil structure. This structure is stabilized by interchain hydrophobic interactions between leucine residues at positions "a" and "d" of a repeating heptad sequence. To determine the positional effects of interchain hydrophobic interactions on the stability of the coiled-coil, a single leucine residue in each chain at position "a" (9, 16, 23, 30) and "d" (5, 12, 19, 26, 33) was systematically replaced by an alanine. All these proteins formed two-stranded alpha-helical coiled-coils in benign conditions (0.05 M phosphate, 0.1 M KCl, pH 7). The stability of each mutant protein was determined by guanidine hydrochloride denaturation experiments, where the decrease in ellipticity at 220 nm was monitored by circular dichroism. The single alanine replacements of a leucine residue at hydrophobic positions a and/or d are all shown to destabilize the coiled-coil structure. The non-equivalent hydrophobic positions a and d make an equivalent contribution to protein stability along the majority of the coiled-coil structure (positions 9-30). The small decrease in coiled-coil stability caused by Leu----Ala substitution at either ends of the coiled-coil suggested that the Leu-Leu hydrophobic interactions are less important at the ends of the coiled-coil and the ends of the coiled-coil are more flexible. Analysis of the difference between the ellipticity in benign buffer and in 50% trifluoroethanol (delta theta 220) and the slope term from a plot of the free energy of unfolding versus guanidine hydrochloride concentration also supported the conclusion that the leucine residues at the ends of the coiled-coil are much less buried than in the middle section of the coiled-coil.

Amino Acid Sequence↗

PubMed1992

The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.

Amino Acid Sequence↗

PubMed1991

Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins.

The performance of a silica-based C8 packing was compared with that of a less hydrophobic, silica-based cyanopropyl (CN) packing during their application to reversed-phase high-performance liquid chromatography (linear trifluoroacetic acid-water to trifluoroacetic acid-acetonitrile gradients) of peptides and proteins. It was found that: (1) the CN column showed excellent selectivity for peptides which varied widely in hydrophobicity and peptide chain length; (2) peptides which could not be resolved easily on the C8 column were widely separated on the CN column; (3) certain mixtures of peptides and small organic molecules which could not be resolved on the C8 column were completely separated on the CN column; (4) impurities arising from solid-phase peptide synthesis were resolved by a wide margin on the CN column, unlike on the C8 column, where these compounds were eluted very close to the peptide product of interest: and (5) specific protein mixtures exhibited superior resolution and peak shape on the CN column compared with the C8 column. The results clearly demonstrate the effectiveness of employing stationary phases of different selectivities (as opposed to the more common optimization protocol of manipulating the mobile phase) for specific peptide and protein applications, an approach underestimated in the past.

Amino Acid Sequence↗

PubMed1989

Reversed-phase chromatographic method development for peptide separations using the computer simulation program ProDigest-LC.

A computer program, ProDigest-LC, has been developed that assists scientists in devising methods of size-exclusion, cation-exchange and reversed-phase high-performance liquid chromatography for the analytical separation and purification of biologically active peptides and peptide fragments from enzymatic and chemical digests of proteins. ProDigest-LC accurately predicts the retention behaviour of peptides of known composition, containing 2-50 amino acid residues, and simulates the elution profiles in all three modes of chromatography. In addition, ProDigest-LC is a user-friendly program, designed as a teaching aid for both students and researchers in selecting the correct conditions for chromatography, that is, the mode of chromatography, column selection and mobile-phase selection, and has the ability to examine the effects of gradient-rate, flow-rate and sample size on the separation. The simulation capabilities of ProDigest-LC as they apply to the reversed-phase chromatography of peptides were examined. The development of the reversed-phase simulation features of the program is described, stressing the importance of peptide standards in the development, testing and practical use of ProDigest-LC. The ease of use of the program is clearly demonstrated by presenting a step-by-step procedure to produce several of the simulations illustrated in the paper. The predictive accuracy of the program was rigorously tested by its application to retention time prediction, at different gradient-rates and flow-rates, for a sample mixture containing peptides exhibiting a wide range of size (11-50 residues), charge (+1 to +8 net charge), hydrophobicity and conformation (random coil to considerable alpha-helical structure). The excellent accuracy of these peptide retention time predictions complemented the successful simulation (in terms of peptide retention times, peptide resolution, peak heights and peak widths) of the effects of gradient-rate and flow-rate on the elution profile of a mixture of closely related peptide analogues.

Chromatography, High Pressure Liquid↗

PubMed1989

Correlation of protein retention times in reversed-phase chromatography with polypeptide chain length and hydrophobicity.

The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time behaviour on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption holds up well enough for small peptides (up to ca. 15 residues), it is now recognized that polypeptide chain length must be taken into account when attempting to equate retention time behaviour of larger peptides and proteins with their overall hydrophobicity. In the present study, we have examined the reversed-phase retention behaviour of 19 proteins of known sequence on stationary phases of varying hydrophobicity and ligand density. From the observed protein retention behaviour on C4, C8 and C18 stationary phases under gradient elution conditions, we have been able to correlate the observed retention times of proteins ranging in molecular weight from 3500 to 32,000 dalton and in chain length from 30 to 300 residues with their overall hydrophobicity (based on retention parameters derived from small peptides) and the number of residues in the polypeptide chain. The retention behaviour of the proteins on the C4, C8 and C18 columns was also compared to that obtained on supports containing lower ligand densities (phenyl ligands). The maintenance of native or partially folded protein conformation on the phenyl columns, resulting in lower retention times than would be expected for fully denatured proteins, underlined the importance of efficient protein denaturation for satisfactory correlation of protein retention times with protein hydrophobicity. In addition, the effectiveness of increasing temperature and/or ligand density of the stationary phase in denaturing proteins was also demonstrated.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Relationship of sidechain hydrophobicity and alpha-helical propensity on the stability of the single-stranded amphipathic alpha-helix.

The aim of the present investigation is to determine the effect of alpha-helical propensity and sidechain hydrophobicity on the stability of amphipathic alpha-helices. Accordingly, a series of 18-residue amphipathic alpha-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic alpha-helix. In these experiments, all three parameters (sidechain hydrophobicity, alpha-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in alpha-helical propensity between the most (Ala) and the least (Gly) alpha-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3 degrees C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and alpha-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic alpha-helix. More importantly, the combined effects of alpha-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with alpha-helix stability. These results suggest that both alpha-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of alpha-helical proteins with the desired stability.

Amino Acids↗

PubMedDate not supplied

Design, synthesis and structural characterization of model heterodimeric coiled-coil proteins.

We report the design and synthesis of model heterodimeric coiled-coil proteins and the packing contribution of interchain hetero-hydrophobic side-chains to coiled-coil stability. The heterodimeric coiled-coils are obtained by oxidizing two 35-residue polypeptide chains, each containing a cysteine residue at position 2 and differing in amino acid sequences in the hydrophobic positions ("a" and "d") responsible for the formation and stabilization of the coiled-coil. In each peptide, a single Ala residue was substituted for Leu at position "a" or "d". The formation and stability of heterodimeric coiled-coils were investigated by circular dichroism studies in the presence and absence of guanidine hydrochloride and compared to the corresponding homodimeric coiled-coils. The coiled-coil proteins with an Ala substitution at position "a" were less stable than those with an Ala substitution at position "d" in both the homodimeric (Ala-Ala interchain interactions) and heterodimeric (Leu-Ala interchain interactions ) coiled-coils. The 70-residue disulfide bridged peptides (homo- and heterodimeric coiled-coils) can be readily separated by reversed-phase chromatography (RPC) even though they have identical amino acid compositions as well as in the hydrophobic "a" and "d" positions. The elution of the 70-residue peptides prior to their corresponding 35-residue monomers suggests that these proteins are retaining a large portion of their coiled-coil structure during RPC at pH2 and their retention behavior correlates with protein stability.

Amino Acid Sequence↗

PubMedDate not supplied

Effect of preferred binding domains on peptide retention behavior in reversed-phase chromatography: amphipathic alpha-helices.

A nonpolar environment, such as the hydrophobic stationary phase of a reversed-phase chromatographic packing, may induce helical structures in potentially helical molecules. If a molecule becomes helical on binding and contains a preferred binding domain, as in the case of an amphipathic helix, then some residues may not be contributing to the same extent to the overall hydrophobicity of the peptide. Amphipathic alpha-helical structures may play an important role in protein folding and the interaction of amphipathic alpha-helices with a hydrophobic surface during RPC is likely to be a good mimic of their hydrophobic interactions with other hydrophobic regions in the folded protein. We have designed and synthesized two sets of model peptides of 7, 14, 21, 28 and 35 residues having the same composition but different sequences (Ac-Lys-Cys-Ala-Glu-Gly-Glu-Leu-[Lys-Leu-Glu-Ala-Gly-Glu-Leu]n-amide and Ac-Lys-Cys-Ala-Glu-Leu-Glu-Gly-[Lys-Leu-Glu-Ala-Leu-Glu-Gly]n-amide, where n = 1-4). Circular dichroism studies demonstrated that both sets of peptides had a high potential to form alpha-helical structure in a nonpolar environment, one set representing amphipathic alpha-helical structures and the other set representing non-amphipathic alpha-helical structures. Size-exclusion chromatography confirmed that all of the peptides in both sets were monomeric when bound to a reversed-phase matrix and also under the conditions used for circular dichroism measurements. Peptides with the same amino acid composition and similar secondary structure could be separated by reversed-phase chromatography. The difference in retention time between peptides of the same length increased with the peptide chain length, ranging from a difference of 2.9 min on a C8 column for the two 14-residue peptides up to a maximum difference of 7.3 min for the 35-residue peptides. From the observed and predicted retention times of these two sets of peptides during reversed-phase chromatography, we have demonstrated that it is possible not only to predict the retention behavior of amphipathic alpha-helices during reversed-phase chromatography, but also to deduce the presence of amphipathic alpha-helical structure in peptides based upon their retention data. If from studies such as these we are eventually able to predict, from only amino acid sequence information, the secondary structure of a peptide on binding to a hydrophobic matrix, we may be able to extrapolate this predictive facility to the conformation of the same sequence in larger polypeptides or proteins.

Amino Acid Sequence↗