Total Chemical Synthesis of a 27 kDa TASP Protein Derived from the

channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthe...
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Bioconjugate Chem. 2002, 13, 474−480

Total Chemical Synthesis of a 27 kDa TASP Protein Derived from the MscL Ion Channel of M. tuberculosis by Ketoxime-Forming Ligation Gerd G. Kochendoerfer,* Janette M. Tack, and Sonya Cressman Gryphon Sciences, 250 East Grand Avenue, Suite 90, South San Francisco, California 94080. Received December 19, 2001; Revised Manuscript Received February 19, 2002

A 27-kDa TASP protein, T5Msc(103-151), that was derived from the cytoplasmic domain (amino acid residues 103-151) of the MscL ion channel of M. tuberculosis was synthesized by ketoxime-forming chemoselective ligation between a template molecule carrying five pyruvic acid groups, and linear channel peptides carrying one aminooxyacetic acid group. Ketoxime-forming ligation provided for highly efficient assembly of this large totally synthetic protein construct with yields >90% with modest excess (1.5×) of the aminooxy peptide. Formation of the desired TASP molecule was confirmed by SDSPAGE analysis and MALDI mass spectrometry. The effect of template attachment on the structure of the peptides constituting the TASP was assessed by circular dichroism spectroscopy. Attachment of the peptides to the topological template induces predominantly helical secondary structure, whereas an analogous peptide that did not bear an aminooxy group, MscL(103-151), does not exhibit significant secondary structure at pH 7 and is found to be monomeric in concentrations up to 65 µM. This observation can be explained by entropic destabilization of the unfolded state of T5Msc(103-151) due to the attachment to the template and the resulting loss of degrees of freedom. Pyruvic acid-based ketoxime-forming chemoselective ligation may thus prove to be a useful tool for the assembly of large, non-native protein constructs and their biophysical study.

INTRODUCTION

The molecular mechanisms underlying the folding and oligomeric assembly of membrane proteins are not fully understood (1, 2). Membrane proteins are thought to be composed of distinct protein domains that fold independently prior to assembly of the full-length protein (1, 3). For that reason, the study of folding and assembly of small membrane proteins, in particular small ion channels, is useful to understand membrane protein folding on an experimental and theoretical level. Recently, we introduced the application of chemical ligation to the total synthesis of membrane proteins. For instance, we showed that both the cytoplasmic and the membrane-spanning domains of totally synthetic influenza A M2 protein help to stabilize the tetrameric assembly of this protein (4). The MscL (mechanosensitive channel large)1 of M. tuberculosis in conjunction with its known crystal structure (5) provides another interesting system to understand the * To whom correspondence should be addressed. E-mail [email protected]; phone (650) 360 1418; fax (650) 952 3055. 1 Abbreviations: Aoaa, aminooxyacetic acid; CD circular dichroism, DCM dichloromethane; DIEA, N,N-diisopropylethylamine; DIC, diisopropycarbodiimide; DMF, dimethylformamide; ESI/MS, electron spray ionization mass spectrometry; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; MALDI-TOF, matrix-assisted laser desorption/ ionization time-of-flight; MscL, mechanosensitive channel large; Mtt, 4-methyltrityl; NHS, N-hydroxysuccinimide; RP-HPLC, reverse phase high performance liquid chromatography;S-DVB, styrene-divinylbenzene; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography; SPPS, solid-phase peptide synthesis; TASP, template-assembled synthetic protein; TM, transmembrane; TFA, trifluoroacetic acid.

assembly of membrane proteins. MscL is one of several channels constituting the stress response of the bacterium and allows the efflux of ions and small molecules in response to osmotic stress (6-8). The crystal structure of the 151-amino acid full-length channel reveals two membrane-spanning helices that are arranged as a homopentamer (5). In addition, this structure indicates that the cytoplasmic residues 103-115-fold into a homopentameric helical pore outside the putative membranespanning region (see Figure 1) (5). As pointed out by the authors, the crystals are disordered beyond residue 115 in the solved structure, and the crystals were obtained at pH 3.5 which casts doubt about the relevance of this structural motif at physiological pH. When performing CD (circular dichroism) measurements on a peptide derived from the C-terminal domain (amino acid residues 103-151) of MscL, we observed that this peptide did not display any secondary structural feature in aqueous solution under neutral conditions, but rather displayed predominant random-coil structure (see below). We hypothesized that the folding of the Cterminal domain of MscL was partially driven by the attachment of the peptides to the topological template formed after the transmembrane domains assemble into the helical pore. Design. In the TASP (template-assembled synthetic protein) approach pioneered by Mutter and co-workers (9-11), peptide building blocks are covalently linked to a topological template in order to direct folding toward a particular three-dimensional packing arrangement and thus mimicking some of the structural and functional properties of native proteins. To test our hypothesis, we designed a TASP that grafts five copies of the C-terminal peptide of MscL (amino acid residues 103-151) onto a peptidic template to mimic the situation in the full-length

10.1021/bc010128l CCC: $22.00 © 2002 American Chemical Society Published on Web 04/09/2002

TASP by Ketoxime-Forming Ligation

Figure 1. Total chemical synthesis of T5Msc(103-151): The channel peptide E102(Aoaa) Msc(102-151) is attached to the linear template peptide by chemoselective oxime forming ligation. The inset shows the crystal structure of MscL of M. tuberculosis (PDB access code: 1MSL, graphics program: deep view). Amino acid residues 103-115 which comprise the N-terminus of the peptides making up T5Msc(103-151) are coded in red.

protein (see Figure 1). The amino acid sequence of the template is identical to the sequence employed by Montal and co-workers (12) to build a pentameric ion channel TASP. The wild-type like activity of this construct demonstrated that nicotinic acetylcholine receptors assemble to form a pentamer, a finding borne out by electron microscopy and crystallographic analysis (1315). We decided to construct a TASP that spans the entire C-terminal domain (starting in the helical region colored in red in Figure 1) beyond the region resolved in the crystal structure to enable future structural work on this protein domain. Our prediction was that attachment of the C-terminal MscL peptide to such a topological template would be sufficient to fold the peptides into a helical pore as observed in the crystal structure. Synthetic Strategy. The TASP design target represents a formidable synthetic challenge due to its large size and complexity. Previously, TASPs have been synthesized either directly by SPPS (solid-phase peptide synthesis) (16, 17) or by independent SPPS of the template and the peptide building blocks, followed by chemoselective ligation or segment condensation of the peptide building blocks to the template (18-29). The large size of the target protein precludes the direct assembly by SPPS due to the large expected number of side-products stemming from the assembly of 256 amino acids in one covalent construct. Therefore, chemoselective ligation is preferred because of the ease of synthesis and purification of unprotected synthetic peptides of ∼50 amino acid residue length, followed by the assembly of these segments into the full-length construct. The challenge was to identify a ligation chemistry that yields complete reaction due to the difficulty in chromatographically

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separating the very similar intermediate trimer and tetramer species from the desired pentameric species. Chemoselective linkage chemistries employed previously for TASP assembly included (a) thioether formation between a thio acid or thiol group and a haloalkyl group (19, 24, 28) or a thiol group and a maleimide group (22, 25, 27), (b) aldoxime formation between an aldehyde and an aminooxy group (18, 20, 21, 23, 29) and reductive amination (26). Initially, we attempted to form the TASP by reaction of a pentamaleimide or a pentaiodoacetyl template with a MscL(103-151) peptide containing an N-terminal cysteine. However, significant amounts (>30%) of dimer, trimer, and tetramer reaction intermediates remaining after completion of the reaction prevented the purification of large quantities of pure material, even in the presence of a 3-fold excess of peptide relative to template. We then considered ketoxime formation for the TASP (to avoid any additional template oxidation/deprotection steps prior to the oximation reaction which are necessary for aldoxime formation). In the literature, levulinic acid and its derivatives are the preferred ketones for chemoselective ligation of nucleophile-bearing groups to peptides and proteins (30-34). However, we and others (33) experienced incomplete reactions using a levulinic acid-based template for attachments of multiple peptides. In this paper, we report a strategy for assembly of TASPs that involves the straightforward, efficient, and complete formation of a ketoxime bond between an aminooxy group on a linear peptide synthesized by Boc chemistry and a pentapyruvic acid bearing template synthesized by Fmoc chemistry. Oxime-forming ligation of these constructs not only provides for efficient assembly of the desired 27 kDa TASP protein, but may have general usefulness for the ligation of synthetic and recombinant peptides. EXPERIMENTAL PROCEDURES

Boc-protected amino acids were obtained from Midwest Biotech (Fishers, IN) and Fmoc-protected amino acids were obtained from Novabiochem (San Diego, CA). Trifluoroacetic acid was obtained from Halocarbon (River Edge, NJ). DIEA (N,N-diisopropylethylamine) was obtained from Applied Biosystems (Foster City, CA). HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) was obtained from Spectrum (Gardena, CA). Carbonyl-free acetonitrile was obtained from Burdick & Jackson (Gardena, CA). Other chemicals were from Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI) and were used as received. Peptide Synthesis and Purification. The sequence of the E102(Aoaa) Msc (102-151) peptide in single-letter code is (Aoaa)-VEQPGDTQ VVLLTEIRDL LAQTNGDSPG RHGGRGTPSP TDGPRASTES Q. The peptide was synthesized at a 0.2 mmol scale on a custom-modified Applied Biosystems 433A peptide synthesizer. The peptide was synthesized on an S-DVB (stryrene-divinylbenzene) resin carrying a -OCH2-PAM linker (Applied Biosystem, Foster City, CA) using an in situ neutralization protocol for machine-assisted Boc (tert-butoxycarbonyl) chemistry (35). Side-chain protecting groups were: Arg(Tos), Asp(OChx), Asn(Xan), Glu(OChx), His(DNP), Lys-(2ClZ), Ser(Bzl), Thr(Bzl), and Tyr(2BrZ). DMF (N,Ndimethylformamide) and DCM (dichloromethane) were HPLC grade and used as received. After the final amino acid coupling, the terminal Boc protecting group was removed by treatment with 100% TFA, followed by neutralization with 10% DIEA in DMF for 1 min. A 2 mmol amount of Boc-aminooxyacetic acid was added to

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the resin after activation with 2 mmol of DIC (diisopropylcarbodiimide) and 2 mmol NHS (N-hydroxysuccinimide) in 3 mL DMF for 30 min. The terminal Boc protecting group was then removed by treatment with 100% TFA for 2 × 1 min. The peptide was deprotected and simultaneously cleaved from the resin support using HF/pcresol according to standard Boc-chemistry procedures (35). The peptide was purified by preparative C-4 RPHPLC with a linear gradient of 20 to 40% Buffer B (acetonitrile containing 0.08% TFA) versus 0.1% aqueous TFA in 80 min. The sequence of the Msc (103-151) peptide in singleletter code is VEQPGDTQ VVLLTEIRDL LAQTNGDSPG RHGGRGTPSP TDGPRASTES Q. It was prepared analogously except for the addition of aminooxyacetic acid. The sequence of the linear template is K(Mtt)AK(Mtt)K(Boc)K(Mtt)PGK(Mtt)E(OtBu)K(Mtt)G. The template was synthesized manually using Fmoc chemistry (36) on an S-DVB resin carrying a Rink linker (Novabiochem, San Diego, CA). For a synthesis on a 0.2 mmol scale, 2.1 mmol of amino acid was dissolved in 3.8 mL of DMF containing 1.9 mmol of HBTU and 1 mL of DIEA and activated for 3 min. Coupling was performed for 20 min per cycle, and deprotection was performed with 2 × 3 min batch washes with 20% piperidine in DMF. After the last amino acid coupling step, the Fmoc protecting group was not removed. The Mtt side-chain protecting group was removed by multiple treatments with 2% TFA in DCM. After neutralization with 10% DIEA in DMF, 1 mmol of pyruvic acid was coupled to the resin after activation with 1 mmol of DIC and 1 mmol of NHS in DCM for 45 min. The template was purified by C-4 RPHPLC with a linear gradient of 15-35% Buffer B versus 0.1% aqueous TFA in 60 min. Fractions containing the desired material were identified by ESI/MS, frozen, and lyophilized. Analytical RP-HPLC. Analytical RP-HPLC was performed on a Vydac C-4 analytical column (5 µm particle size, 0.46 cm × 15 cm), using a Hewlett-Packard Model 1100 quaternary pump high-pressure mixing system with 214 and 280 nm UV detection. Oxime-Forming Chemoselective Ligation. A 1.5fold molar excess (aminooxy acetyl) peptide to pyruvic acid group on the template) of E102(Aoaa) Msc (102151) was added to a solution of the template in 50% aqueous acetonitrile containing 0.09% TFA. After 15 min reaction time, solvents were evaporated to dryness on a speedvac to drive the reaction to completion, and the dry mix was redissolved in 50% aqueous acetonitrile containing 0.09% TFA. The N-terminal Fmoc-protecting group on the template was removed by addition of a 3-fold excess of 6 M guanidinium chloride, 100 mM acetate, pH 4 buffer, and an equivalent volume of piperidine (final concentration 20%) for 20 min. The solution was acidified with glacial acetic acid, and reactants and products were separated by preparative reverse-phase HPLC with a linear gradient of 20 to 48% Buffer B (acetonitrile containing 0.08% TFA) versus 0.1% aqueous TFA in 80 min. Fractions containing the desired material were identified by SDS-PAGE, frozen, and lyophilized. Mass Spectrometry. T5Msc(103-151) was dissolved in a saturated solution of R-cyano-4-hydroxycinnamic acid in 50% aqueous acetonitrile/0.09% TFA acid. A 1 µL aliquot was deposited directly on the sample slide and allowed to air-dry. The sample preparations were analyzed using a Ciphergen Biosystems Massphoresis System time-of-flight mass spectrometer. The spectra were externally calibrated using horseradish peroxidase (MW ) 43240 Da) and represent an average of 50 shots.

Kochendoerfer et al.

Protein Folding. To induce folding, T5Msc(103-151) and the Msc (103-151) peptide were dissolved at a concentration of 1 mg/mL in 6 M guanidinium hydrochloride buffer containing 200 mM H2PO4- (counterion sodium), pH 7, respectively. The peptides were dialyzed stepwise O/N against 100× volume into 20 mM H2PO4(counterion sodium), pH 7, containing 100 mM NaCl, and finally 20 mM H2PO4- ((counterion sodium), pH 7. For folding at low pH, the final buffer was 20 mM glycine (counterion chloride), pH 4. Circular Dichroism Spectrometry. CD spectra were acquired with a JASCO-J700 CD spectrometer at 20 °C between 190 and 250 nm in 0.4 nm increments (bandwidth: 1 nm, dwell-time: 2 s, averaged over three acquisitions). Quantitative secondary structure modeling was performed by least-squares fitting to a set of proteins of known structure using the CDsstr and Continll algorithms as implemented by the CDPRO software package (http://lamar.colostate.edu/∼sreeram/CDPRO). The basis set employed for modeling the linear peptide also included several unfolded proteins, since the fits in the absence of unfolded proteins in the basis set were unsatisfactory. The Selcon3 algorithm implemented in the same software package did yield significantly worse fits as indicated by more than four times higher RMSD values (experimental - calculated) and was therefore discarded. However, the determined percentage of helicity and disordered structure was comparable. RESULTS

The channel peptide E102(Aoaa) MscL (102-151) was assembled using Boc chemistry SPPS following standard procedures (35). After chain assembly, an aminooxy acetic acid group was coupled to the N-terminus of the peptide to serve as chemoselective linker. The peptide was deprotected, cleaved, and purified using standard methods (35). For structural comparison, an analogous peptide that did not bear an aminooxy group (MscL(103-151) was synthesized. The template peptide (12) was synthesized by SPPS following the Fmoc strategy as described in the methods section (36). After chain assembly, pyruvic acid was coupled to five specific side-chains to serve as chemoselective ligation linkers. The TASP T5Msc(103-155) was assembled by joining five unprotected, purified channel peptides to the purified template peptide through a chemoselective ketoxime-forming ligation (See Scheme 1). A Scheme 1. Ketoxime-Forming Ligation by Reaction of a Pyruvic Acid Bearing Peptide (left) with an Aminooxyacetic Acid Bearing Peptide (right). The Newly Formed Oxime Bond Is Stabilized by Electron Resonance

TASP by Ketoxime-Forming Ligation

Figure 2. Analysis of oxime-forming ligation: Left: RP-HPLC traces (detected at 214 nm) monitoring the oxime-forming ligation reaction. Analytical reversed-phase HPLC was performed on aliquots with a linear gradient of 5-65% Buffer B (acetonitrile containing 0.08% TFA) versus 0.1% aqueous TFA over 24 min on a Vydac C-4 analytical column. Top: RP-HPLC trace after injection of an aliquot of the reactant peptide E102(Aoaa)Msc(102-151). Bottom: Equivalent RP HPLC trace after the oximation reaction. Right: NuPAGE Bis-Tris (Mes) SDSPAGE gel (4-12%) monitoring the synthesis and purification of T5Msc(103-151). Gels were stained for protein with Coomassie blue. Lane 1 + 5: MW Standard. Lane 2: Reactant (E102Aoaa) Msc(102-151). Lane 3: Crude oximation mix. Lane 4: Purified T5Msc(103-151).

1.5-fold molar excess (aminooxy peptide to pyruvic acid unit on the template) of E102(Aoaa) MscL (102-151) was added to a solution of the template in 50% aqueous acetonitrile containing 0.09% TFA. Figure 2 presents HPLC traces monitoring the formation of the target TASP, named T5Msc(103-151). The linear E102(Aoaa) MscL (102-151) peptide exhibits a single dominant peak at 19.0 min. A minor peak at 19.8 min corresponds to an aminooxy-mediated dimer that is intrinsically unreactive and appears after lyophilization of the purified peptide. After the reaction mixture was dried on a Speedvac and redissolved in 50% acetonitrile containing 0.09% TFA, a new peak at 21.2 min appears. The increase of this peak is correlated with a decrease of the reactant peak. To monitor the completeness of the reaction more quantitatively, an SDS-PAGE gel of the reactant, and the product mix after dissolution in SDS sample buffer was obtained. The monomeric starting material in Lane 2 (Figure 2) shows the expected molecular weight of ∼5 kDa. The crude reaction mixture in Lane 3 indicates very high reaction efficiency. In addition to the unreacted starting material and the desired pentamer band at ∼31 kDa, only a weak band for the tetramer (98% complete for each aminooxyacetic-pyruvic acid ketone pair even with the modest excess (1.5-fold) of aminooxy peptide employed. After workup, reactants and products were separated by preparative RP-HPLC as described in the methods section. Fractions containing the desired TASP protein, T5Msc(103-151), were identified by SDS-PAGE, frozen, and lyophilized. Lane 4 in the SDS-PAGE gel in Figure 2 indicates high (>98%) oligomeric purity for the purified pool of T5Msc(103-151). Additional analytical data characterizing purified T5Msc(103-151) is presented in Figure 3. The RP-HPLC trace of the purified TASP protein shows a single

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Figure 3. Analytical characterization of T5Msc(103-151): Top: Reverse-phase HPLC chromatogram of purified T5Msc(103-151) construct. Analytical reverse-phase HPLC was performed with a linear gradient of 5-65% Buffer B (acetonitrile containing 0.08% TFA) versus 0.1% aqueous TFA over 24 min on a Vydac C-4 analytical column. The inset shows a MALDITOF mass spectrum of purified T5Msc(103-151). Peaks originating from doubly protonated species are marked as (2H). Bottom: Size-exclusion HPLC chromatogram of the purified and “folded” T5Msc(103-151) (Top) and Msc(103-151) (Bottom) construct. Analytical SEC HPLC was performed on aliquots using 20 mM H2PO4-, pH 7 containing 100 mM NaCl as mobile phase on a Tosoh-Haas G2000 SWXL column (particle size 5 µM, pore size 125 Å). Size exclusion standards eluted at the following retention times: albumin (69 kDa): 14.9 min; ovalbumin (46 kDa): 15.9 min; chymotrypsin (21 kDa): 21.9 min.

dominant peak with a peptidic purity greater than 95%. Formation of the desired pentameric TASP was verified by MALDI-TOF mass spectrometry. The observed molecular weight of T5Msc(103-151) is 27390 ( 20 Da (theoretical mass ) 27389 (average isotope composition)). The impurity observed in the MALDI spectrum at ∼5 kDa below the target molecular weight is attributed to cleavage of the UV-absorbing oxime bond by the excitation laser as well as possibly a slight channel peptide tetramer impurity. The latter interpretation is confirmed by a clear laser power dependence of the relative intensity of this impurity peak. To induce folding, T5Msc(103-151) and Msc(103-151) were dissolved in denaturing buffer, followed by dialysis into 20 mM H2PO4-, pH 7. Size-exclusion chromatographic analysis (Figure 3, bottom) indicates that the retention time (15.1 min) of “folded” T5Msc(103-151) lies between the retention times of the molecular weightstandards ovalbumin (∼46 kDa) and albumin (69 kDa). The apparent discrepancy of this apparent weight to the real confirmed molecular weight can be rationalized by the expected nonglobular, possibly rodlike shape of the TASP. Rodlike proteins are expected to be more hindered when entering the pores of the size-exclusion resin, thus exhibiting higher apparent molecular weights (37). An alternative explanation is that a significant part of the TASP may be (a) unfolded or only loosely structured, leading to increased apparent molecular weight or (b) completely dimeric. These alternatives, while less likely,

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Kochendoerfer et al.

Figure 4. Left: Circular dichroism spectra of Msc(103-151) (diamonds) and T5Msc(103-151) (squares) in 20 mM H2PO4- (counterion sodium), pH 7. Data fits obtained from the quantitative secondary structure modeling are also shown for the measurements at pH 7 (dashed lines). The concentrations of T5Msc(103-151) and Msc(103-151) were 42 µg/mL (1.5 µM) and 38 µg/mL (7.4 µM), respectively. Right: Circular dichroism spectra of Msc(103-151) (diamonds) and T5Msc(103-151) (squares) in 20 mM glycine (counterion chloride), pH 4. The concentrations of T5Msc(103-151) and Msc(103-151) were 58 µg/mL (2.1 µM) and 84 µg/mL (16.5 µM), respectively.

will be addressed in further studies. In summary, it appears that MscL (103-151) is predominantly monomeric (>90%) with a dimeric species (retention time 13.3 min) representing the major additional population under our assay conditions up the highest concentration (∼65 µM) employed in the CD analysis. To assess folding of T5Msc(103-151), CD spectra were acquired of the TASP and MscL (103-151), respectively, in aqueous buffer. The final buffers were 20 mM H2PO4-, pH 7, and 20 mM glycine buffer, pH 4, respectively. Figure 4 presents the CD spectrum of the linear Msc (103-151), and the CD spectrum of T5Msc(103-151), respectively, under comparable total peptide concentrations in 20 mM H2PO4-, pH 7. Inspection of the CD trace of Msc(103-151) indicates predominant random coil structure at pH 7. A significant increase in helicity is observed after lowering the pH to 4 for this peptide. SEC (size-exclusion chromatography) reveals that the peptide is monomeric at pH 4 as well (data not shown). Subsequently, the concentration dependence of the helical content was determined since for a pentameric oligomer one expects a strong dependence of the oligomeric state on the concentration. The respective CD spectra do not change significantly over the range from 7.4 to 66 µM for the peptide at pH 7 and 3.5 to 65 µM for the peptide at pH 4. Thus, peptide MscL (103-151) is intrinsically helical in the monomeric state at low pH, but predominantly in a random coil conformation at physiological pH. By contrast, T5Msc(103-151) exhibits a CD trace at both pH 7 as well as pH 4 typically observed for proteins with a high helical content and shows a slight decrease in helicity at pH 4 relative to pH 7. The CD spectra of T5Msc(103-151) and Msc(103-151) at pH 4 are virtually superimposable at higher wavelengths (intrinsic absorption of UV light, most likely by the oxime bond, precluded acquisition of data beyond this region). Quantitative secondary structure modeling of the data obtained at pH 7 revealed that whereas the linear synthetic peptide exhibits ∼15% helical structure and is primarily comprised of disordered structure (61-73%), assembly of the peptides on the peptide template increases the helical content to 60-67%, with only 20% disordered structure. Whereas these numbers should not be taken literally due to the limited accuracy of quantitative CD analysis, they do confirm the trend observed by visual inspection. A small percentage of β-sheet structure (