Photo-control of peptide helix content by an azobenzene cross-linker: steric interactions with underlying residues are not critical

Janet R. Kumita1, Daniel G. Flint2, Oliver S. Smart2 and G.Andrew Woolley1,3

1 Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto M5S 3H6, Canada and 2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Photo-control of protein conformation could prove useful for probing function in diverse biological systems. Recently, we reported photo-switching of helix content in a short peptide containing an azobenzene cross-linker between cysteine residues at positions i and i + 7 in the sequence. In the original sequence, underlying residues at positionsi + 3 and i + 4 were made bulky as preliminary modelling suggested that this would enhance photo-control of helix content. To test this hypothesis, peptides with Val, Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 were synthesized, cross-linked and characterized. Before cross-linking, the peptides show distinct conformational behaviours: two with differing helix/coil mixtures whereas the other has a circular dichroism (CD) spectrum characteristic of ß-sheet and a tendency to aggregate. However, upon cross-linking the peptides have very similar CD spectra: predominantly random coil in the dark but predominantly helical upon irradiation. These results refute the original hypothesis. Steric interactions between the linker and underlying residues do not appear to be critical for photo-switching behaviour. When the cross-linking bridge is lengthened by replacing the i, i + 7 cysteine residues with homocysteine, a lower degree of photo-control of helicity is observed. Furthermore, a non-cross-linking version of the azobenzene reagent is shown not to produce any photo-control of helicity. We conclude that the intramolecular cross-link is essential for photo-switching and that it should be applicable to a wide range of peptides and proteins.

Keywords: {alpha}-helix coil/conformation/light-induced/photo-control/photo-isomerization


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A variety of approaches for controlling helix stability have been described (Bodkin and Goodfellow, 1995Go; Rohl and Baldwin, 1998Go; Miller et al., 2001Go). One general strategy involves optimization of peptide primary sequence to maximize helix content. Substantial experimental and computational work aimed at determining intrinsic helical propensities, side chain–side chain interaction energies, electrostatic interactions, main chain–main chain hydrogen bonds and capping effects has allowed the accurate prediction of helix propensities in monomeric peptides (Lacroix et al., 1998Go; Fisinger et al., 2001Go). Alternative strategies for helix stabilization include the incorporation of metal chelates (Ghadiri and Fernholz, 1990Go; Ruan et al., 1990Go), intramolecular disulfide linkages (Pease et al., 1990Go; Jackson et al., 1991Go) and intramolecular side chain tethers (Osapay and Taylor, 1992Go; Luo et al., 1994Go; Phelan et al., 1997Go; Yu and Taylor, 1999Go).

Recently, we reported a reversible means of controlling helix stability that involved the incorporation of a photo-isomerizable azobenzene cross-linking reagent into an engineered peptide system (Kumita et al., 2000Go). Optical control of peptide/protein conformation provides a new biochemical tool for probing protein function in diverse systems. Moroder, Chmielewski and colleagues have incorporated azobenzene groups directly into the peptide backbone (Ulysse et al., 1995Go; Behrendt et al., 1999Go; Renner et al., 2000aGo,bGo) and have studied the conformational effects of photo-isomerization in these systems. Conformational effects of azobenzene containing side chains have also been studied (Cerpa et al., 1996Go; Liu et al., 1997Go; Zhang et al., 1999Go).

The photo-controlled helical peptide that we previously reported was prepared by combining a thiol-reactive azobenzene cross-linker with a peptide containing Cys residues spaced at positions i, i + 7 in the sequence (Kumita et al., 2000Go). This design (designated JRK-{ValAib}-X) was based on molecular modelling studies that indicated the azobenzene cross-linker in the trans conformation was too long to allow the i, i + 7 residues and intervening residues to adopt an {alpha}-helix conformation. When the cross-linker was in the cis conformation, its length better matched the spacing of i, i + 7 residues in an ideal {alpha}-helix and it would thus be expected to act as an entropic helix stabilizer.

The residues i + 3 and i + 4 lie beneath the azobenzene linker joining the Cys residues at i and i + 7 in the (helical) cross-linked peptide sequence. The original modelling method employed suggested that, if bulky side chains were incorporated at positions i + 3 and i + 4, steric interactions between these side chains and the cross-linker would lead to further helix destabilization of the trans form of JRK-{ValAib}-X. The side chains of the residues at i + 3 and i + 4 were not proposed to interact sterically with the cross-linker in the cis conformation (Kumita et al., 2000Go). These considerations led to the inclusion of Val (i + 3) and Aib ({alpha}-aminoisobutyric acid) (i + 4) in the JRK-{ValAib}-X sequence. The bulky ß-branched side chain of Val and the pro-R methyl group of Aib were proposed to interact directly with the trans azobenzene group if the peptide were confined to a helical conformation. In addition, since the Aib residue has an {alpha}-helix enhancing effect in peptides (Kaul and Balaram, 1999Go), it could be expected to offset any reduction in helix propensity caused by the helix destabilizing ß-branched Val residue. Circular dichroism (CD) spectra of JRK-{ValAib}-X indicated that the helix content was increased substantially upon photo-isomerization of the linker from trans to cis and decreased upon cis to trans isomerization. However, it was not clear to what extent steric interactions between the Val (i + 3) and Aib (i + 4) residues and the azobenzene linker were responsible for this effect. Importantly, if the inclusion of Aib (a non-coded amino acid) were not critical, then this strategy for photo-control of helix stability could, in principle, be applied to a wide range of expressed proteins with appropriately spaced Cys residues.

In this paper we address the role of steric interactions between positions i + 3, i + 4 and the linker in the photo-switching behaviour of this peptide system. The i, i + 7 spacing of the linker was maintained and targeted residue substitutions were made. Bulkier [Ile (i + 3), Aib (i + 4)] and less bulky [Ala (i + 3), Ala (i + 4)] were tested. The effect of replacing Cys residues at i, i + 7 with the more flexible homocysteine (hCys) was also tested. Together with the experimental work, improved computational methods were developed to assess more accurately the role of steric interactions in the conformational dynamics of these systems. Finally, a non-cross-linking version of the azobenzene modifier was tested to confirm the importance of the intramolecular cross-link.

We report here that the helix content of all the cross-linked peptide variants can be photo-controlled reversibly and with approximately the same degree of structural change as the original system. These results imply that the photo-isomerizable cross-linker strategy can be used to affect helix stability in a wide range of synthetic and/or expressed peptides and proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synthesis of the azobenzene cross-linking reagent

The azobenzene cross-linking reagent containing two cysteine-reactive iodoacetamide groups (structure 1) was synthesized as reported previously (Kumita et al., 2000Go).

Synthesis of the azobenzene modifying reagent

4-Phenylazoaniline (300 mg, 1.52 mmol) (Sigma-Aldrich Canada) was dissolved in anhydrous THF (5 ml) and stirred under nitrogen for 15 min protected from light. Four equivalents of TEA (841 ml, 6.08 mmol) were added, followed by the dropwise addition of 3 equiv. of chloroacetyl chloride (363 ml, 4.56 mmol). The reaction mixture was filtered and the solvent evaporated. The crude product was purified on a silica gel column (75:25 CHCl3–MeOH). The purified solid was dissolved in acetone (8 µl) and an excess of sodium iodide (30 mmol) was added. The reaction was stirred under nitrogen, protected from light for 18 h. The reaction mixture was filtered to remove NaCl and the solvent was removed to give a brownish–orange solid. The crude product was dissolved in chloroform (3 µl) and aqueous extraction was performed to remove excess NaI. The organic layer was dried over anhydrous sodium sulfate, filtered and dried. 1H NMR and high-resolution electron impact MS confirmed the synthesis of the iodoacetamide azobenzene compound (structure 2). Rf = 0.91 in 75:25 CHCl3–MeOH. 1H NMR (CDCl3, 400 MHz): {delta} 3.92 (s, 2H), 7.40 (m, 3H), 7.7 (d, J = 8.42 Hz, 2H), 7.86 (s, 1H), 7.93 (d, J = 6.50 Hz, 2H), 7.97 p.p.m. (d, J = 8.42, 2H). High-resolution electron ionization MS: observed mass = 365.002926, calculated mass (for C14H12N3OI) = 365.002514.

Peptide synthesis

Standard fluorenylmethoxycarbonyl-based solid-phase peptide synthesis methods were used to prepare all peptides; JRK-{ValAib}, acetyl-EACARVAibAACEAAARQ-NH2; JRK-{IleAib}, acetyl-EACARIAibAACEAAARQ-NH2; JRK-{AlaAla}, acetyl-EACARAAAACEAAARQ-NH2; JRK-{hCys}{ValAib}, acetyl-EAhCARVAibAAhCEAAARQ-NH2. Peptides were constructed on Pal-resin (capacity 0.55 mmol/g) (Advanced ChemTech, Louisville, KY). Coupling used 3 equiv. HATU [O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (Sigma-Aldrich Canada), 6 equiv. DIPEA (N,N-diisopropylethylamine) and 3 equiv. amino acid (Novabiochem, San Diego, CA). Aib-F was synthesized following the procedure of Kaduk et al. (Kaduk et al., 1996Go). Peptides were purified by HPLC [8 µ Apex Presil C18 column (Jones Chromatography, Lakewood, CO)] using a linear gradient from 0 to 50% acetonitrile–H2O (containing 0.1% trifluoroacetic acid) over 40 min for JRK-{ValAib} (eluted at 47% acetonitrile) and a linear gradient from 0 to 80% acetonitrile–H2O (containing 0.1% trifluoroacetic acid) over 45 min for JRK-{IleAib} (eluted at 56% acetonitrile), JRK-{AlaAla} (eluted at 47.1% acetonitrile) and JRK-{hCys}{ValAib} (eluted at 49.4% acetonitrile). The peptide primary structures were confirmed by electrospray ionization MS and amino acid analysis (HSC Biotechnology Service Centre, Toronto): JRK-{ValAib} [observed 1645.5 Da; calculated (C65H112N24O22S2) 1645.9 Da]; JRK-{IleAib} [observed 1659.4 Da; calculated (C66H114N24O22S2) 1659.9 Da]; JRK-{AlaAla} [observed 1604.2 Da; calculated (C62H106N24O22S2) 1603.8 Da]; and JRK-{hCys}{ValAib} [observed 1673.2 Da; calculated (C67H116N24O22S2) 1673.9 Da]. The purity by HPLC was >95%.

Peptide cross-linking and purification

Intramolecular cross-linking of Cys residues by 1 was performed as detailed by Kumita et al. (Kumita et al., 2000Go) for JRK-{ValAib}, JRK-{IleAib} and JRK-{hCys} {ValAib}. The incubation time of the peptide with tris(carboxy-ethyl)phosphine (TCEP) was increased to 3 h to ensure that cysteine residues were in the reduced state. Cross-linked peptides are indicated by an X following the peptide identification. For JRK-{AlaAla}, the original cross-linking procedure was modified owing to the limited solubility of the unmodified peptide. In a total volume of 750 ml of 15.5 mM Tris–HCl buffer (pH 8), uncross-linked JRK-{AlaAla}(0.413 mM), TCEP (0.413 mM) and guanidine hydrochloride (2 M) were combined and incubated for 18 h at room temperature, under nitrogen to ensure the cysteine residues were in their reduced state. To the aqueous solution, 1 ml of DMSO containing 0.46 mmol of cross-linker 1 was added. This solution was stirred for 10 min protected from light and then 46.5 ml of a 10 mM solution of 1 was added to the mixture. This was repeated three times (at 10 min intervals) for a total reagent concentration of 1.2 mM. The reaction mixture was stirred for a further 20 min exposed to ambient light.

Unreacted 1 was removed by Biogel P4 gel-filtration chromatography and peptides were purified by HPLC (Zorbax SB-C18 column) using a linear gradient from 0 to 80% acetonitrile–H2O (containing 0.1% trifluoroacetic acid) over the course of 45 min for JRK-{ValAib}-X (eluted at 43.2% acetonitrile), JRK-{IleAib}-X (eluted at 43.6% acetonitrile), JRK-{AlaAla}-X (eluted at 41.2% acetonitrile) and JRK-{hCys}{ValAib}-X (eluted at 46.4% acetonitrile). The peptide primary structures were confirmed by MALDI-MS and amino acid analysis (HSC Biotechnology Service Centre): JRK-{ValAib}-X [observed 1937.6 Da; calculated (C81H128N28O24S2) 1938.2 Da]; JRK-{IleAib}-X [observed 1952.7 Da; calculated (C82H126N28O24S2) 1952.2 Da]; JRK-{AlaAla}-X [observed 1895.6 Da; calculated (C78H118N28O24S2) 1896.1 Da]; and JRK-{hCys}{ValAib}-X [observed 1967.0 Da; calculated (C83H129N28O24S2) 1967.2 Da].

Reaction of JRK-{ValAib} with the non-cross-linking azobenzene reagent

Reaction of the two cysteine residues in JRK-{ValAib} with compound 2 was performed as follows. In a total volume of 355 µl of 15.5 mM phosphate buffer (pH 8), uncross-linked peptide JRK-{ValAib} (0.89 mM) and TCEP (0.89 mM) were combined and incubated for 1 h at room temperature to ensure that cysteine residues were in their reduced state. To the aqueous solution, 500 µl of DMSO containing an excess of 2 (2.0 mmol) was added and the mixture was stirred for 18 h protected from light and under nitrogen. Unreacted 2 was removed by Biogel P4 gel-filtration chromatography and the modified peptide was purified by HPLC [Zorbax SB-C18 column; 0–80% acetonitrile–H2O (containing 0.1% trifluoroacetic acid) linear gradient over the course of 45 min; elution at 64% acetonitrile]. The peptide primary structure was confirmed by amino acid analysis (HSC Biotechnology Service Centre) and electrospray ionization MS: JRK-{ValAib}-Mod [observed 2119.6 Da; calculated (C93H134N30O24S2) 2120.4 Da].

Molecular modelling

The cross-linker was sketched in SYBYL 6.7 in both the trans and cis isomeric states and optimized in Gaussian 98 (Frisch et al., 1998Go) at the HF level with the 3–21G basis set. The HF/3–21G combination of method and basis set was chosen to be consistent with the later QM/MM optimizations. Theoretical studies of both trans and cis azobenzene (Biswas and Umapathy, 1997Go; Kurita et al., 2000Go) and our own calculations have shown that the ab initio HF method is able to represent well both the geometries and energies of these two isomers. However, none of the common semi-empirical methods [e.g. AM1 used by Kumita et al. (Kumita et al., 2000Go)] offer acceptable representations (Kurita et al., 2000Go). All QM and QM/MM calculations were performed using Gaussian 98 on a DEC ALPHXP1000 workstation with an EV6 processor. For modelling and minimization with SYBYL 6.7, an SGI O2 workstation with an R10000 processor was used.

Peptides were built as {alpha}-helices in SYBYL 6.7. Glu, Arg and Gln residues in the experimental peptides were changed to Ala for the theoretical work to simplify the peptides for modelling and to allow the interactions between the cross-linker and the residues at i + 3 and i + 4 to be examined in detail. The peptides were energy minimized using the all-atom AMBER molecular mechanics (MM) forcefield (Cornell et al., 1995Go).

Once the separate components of cross-linker and peptide had been modelled they were bonded together to construct a model of the cross-linked peptide as completely {alpha}-helical. A systematic study was performed to choose optimal values for the Cys side chain dihedral angles based on a study of dihedral angles (Stapley and Doig, 1997Go) in protein structures and our own energy calculations. The cross-linked peptides were initially subjected to energy minimization with the AMBER forcefield. The cross-linker was kept rigid with Cartesian constraints on all its atoms. This procedure allowed the cross-linker to remain in an energetically relaxed conformation while forcing the peptide to undergo conformational changes to compensate.

The MM minimized cross-linked peptide was then optimized with the ONIOM combined QM/MM representation (Svensson et al., 1996Go). The cross-linker was represented at the ab initio HF level with a 3–21G basis set. Larger basis sets caused SCF convergence problems. Using QM rather than MM for the azobenzene-peptidyl cross-linker, we were able to use an accurate but parameterization-free representation for this part of the molecule. AMBER was used to represent the peptide since this forcefield has to been shown to represent well the conformational properties of proteins. Partial charges for the cross-linker-modified Cys residues were generated using the RESP model (Bayly et al., 1993Go). Atomic partial charges were scaled for a constant dielectric of 10 to represent implicit aqueous solvent effects on electrostatic interactions in the peptide.

The optimized cross-linked peptides were then assessed for the alteration in helicity by DSSP (Kabsch and Sander, 1983Go). The energy classes for {alpha}-helical (i, i + 4) hydrogen bonds were delineated as follows: weak (–0.6 to –1.0 kcal/mol), intermediate (–1.1 to –1.6 kcal/mol) and strong (<=–1.7 kcal/mol). For comparison, DSSP returned an energy of –2.3 kcal/mol for a hydrogen bond in an ideal polyalanine helix optimized with the same scaling of partial charges.

Circular dichroism measurements

Circular dichroism (CD) measurements were performed with a Jasco Model J-710 spectropolarimeter. All measurements were made in a thermostatted quartz cuvette (0.1 cm pathlength). Temperatures were measured using a microprobe directly in the sample cell. All samples were dissolved in 5 mM phosphate buffer (pH 7). Dithiothreitol (DTT) (3 mM) was present in the uncross-linked peptide samples to ensure that cysteine and homocysteine residues were in the reduced form. Spectra reported are averages of three individual experiments of five scans each, with the appropriate background spectrum subtracted unless reported otherwise. A scan speed of 10 nm/min, with a 0.5 nm bandwidth and a 4 s response time, was used. The mean residue weights used for both the uncross-linked and modified peptides were 102.8 (JRK-{ValAib}, JRK-{ValAib}-X and JRK-{ValAib}-Mod), 103.7 (JRK-{IleAib} and JRK-{IleAib}-X), 100.3 (JRK-{AlaAla} and JRK-{AlaAla}-X) and 104.6 (JRK-{hCys}{ValAib} and JRK-{hCys}{ValAib}-X). Helix content was calculated using the simple assumption that 100% helix gives a {theta}222 value of (–40 000)[(n – 4)/n], where n is the number of residues (Kallenbach and Spek, 1998Go).

UV/Vis analysis and photo-isomerization

UV spectra were obtained with a Perkin-Elmer Lambda 2 spectrophotometer using the same thermostatted quartz cell (0.1 cm pathlength) in which CD analysis was performed. Based on the peptide concentration determined by quantitative amino acid analysis for JRK-{ValAib}-X, the molar extinction coefficient for the trans cross-linker was determined to be 28 000 M–1 cm–1 at 367 nm. This extinction coefficient was then used to determine peptide concentrations for all dark-adapted cross-linked peptides directly in the CD cuvette. A molar extinction coefficient of 30 000 M–1 cm–1 at 340 nm was determined for compound 2 and used to determine the concentration of JRK-{ValAib}-Mod.

Photoisomerization was accomplished by irradiating thermostatted peptide solutions with a 70 W metal halide Tri-Lite lamp (World Precision Instruments) coupled to a 370 ± 10 nm (or 340 ± 40 nm for JRK-{ValAIb}-Mod) bandpass filter (Harvard Apparatus Canada). Photoisomerization was complete (as judged by the lack of further changes in UV spectra) in <=5 min.

Determination of cis contents in irradiated cross-linked peptides

Spectra for pure trans and cis forms of JRK-{ValAib}-X were obtained using a Waters 996 photodiode array (PDA) detector coupled to an HPLC system comprised of a Waters 600 series controller and pump (Waters, Milford, MA). The trans and cis isomers of JRK-{ValAib}-X were separated on an Alltima C18 5mm (250 mmx4.6 mm i.d.) column preceded by an Econosil C18 10 mm guard cartridge (Alltech, Guelph, ON). A linear gradient from 20 to 70% A over 10 min followed by 70% A for 5 min (where solvent A was 80% acetonitrile–20% water containing 0.1% trifluoroacetic acid) was used. The retention time for the trans conformation was 13.83 min and that for the cis conformation was 14.27 min. By comparing the peak areas at 310 nm (an isosbestic point), the percentage of each isomer present in the sample could be determined. The total concentration of the sample was calculated based on the molar extinction coefficient for the dark-adapted (trans) form.

The UV spectrum of the cross-linker is essentially unaffected by variations in peptide structure for wavelengths >330 nm. Thus the pure trans and cis spectra determined by the HPLC–PDA experiment were used to determine the percentage of cis peptide obtained after irradiation in each case. The values obtained were: JRK-{ValAib}-X 77%, JRK-{IleAib}-X 68%, JRK-{AlaAla}-X 72% and JRK-{hCys}{ValAib}-X 73%. Using these percentages of cis form observed, theoretical 100% cis CD spectra ({theta}222 nm values in Table IIGo) were calculated using the following equation: {theta}222 nm (100% cis) = {{theta}222 nm (observed after irradiation) – [fraction transx{theta}222 nm (dark-adapted)]}/fraction cis.


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Table II. Helical content of cross-linked peptides
 

    Results
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Peptide sequence variants: uncross-linked

To investigate the importance of the Val (i + 3), Aib (i + 4) residues in the secondary structure change observed with JRK-{ValAib}-X, three peptide variants were synthesized, cross-linked and characterized (Figure 1AGo). Valine was replaced with the bulkier Ile residue in JRK-{IleAib}-X, whereas in JRK-{AlaAla}-X, both Val (i + 3) and Aib (i + 4) were replaced with the less bulky Ala residue. To increase the degree of flexibility between the i and i + 7 {alpha}-carbons and the azobenzene moiety, Cys was replaced with hCys (JRK-{hCys}{ValAib}-X), which contains an extra methylene group between the {alpha}-carbon and the thiol group.



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Fig. 1. Structures of peptide variants and azobenzene reagents. (A) Primary sequence of the cross-linked peptides. AZO refers to the cross-linker (1) after reaction with the two cysteine side chains [or homocysteine (hCys) in JRK-{hCys}{ValAib} (Aib = {alpha}-aminoisobutyric acid)]. (B) Structures of the azobenzene cross-linker (1) and the azobenzene modifying reagent (2).

 
The conformational properties of these sequence variants were first examined in uncross-linked form by CD spectroscopy since single mutations to short peptides can have drastic effects on the degree of {alpha}-helicity (Merutka et al., 1990Go). Each of these samples contained excess DTT to keep thiol groups in their reduced form. The observed mean residue ellipticities and AGADIR-predicted helicity (Munoz and Serrano, 1994Go) are collected in Table IGo.


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Table I. Helix content of unmodified peptides
 
The peptides JRK-{hCys}{ValAib} and JRK-{IleAib} showed higher helical contents than the native sequence (JRK-{ValAib}) although they were only of the order of 40% helical. JRK-{AlaAla} however, displayed a tendency to aggregate and a non-helical CD spectrum (Figure 2AGo).



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Fig. 2. Secondary structure of uncross-linked JRK-{AlaAla}. (A) CD spectrum of JRK-{AlaAla} [50 mM peptide, 5 µM sodium phosphate buffer (pH 7), 3 mM DTT, 10°C]. (B) Model of an antiparallel ß-sheet that may be formed by JRK-{AlaAla}. (i) Side view showing hydrophobic Ala-rich face on the bottom and Glu/Arg residues (space-filling) on the top. (ii) Top view of an antiparallel sheet showing interactions between Glu/Arg residues on adjacent strands. These sheets could further associate via their hydrophobic bottom surfaces. Part (B) was produced using Molscript (Kraulis, 1991Go).

 
Peptide sequence variants: cross-linked

Each of the peptides was treated with the azobenzene cross-linking reagent (1) (Figure 1BGo) as described in the Materials and methods section. Owing to the limited solubility of JRK-{AlaAla}, guanidine hydrochloride had to be included in the reaction mixture. Successful intramolecular cross-linking was achieved in each case, as demonstrated by the presence of singly ionized molecular ion peaks in MALDI mass spectra. Cross-linked peptides are denoted with an X following the peptide identification (e.g. JRK-{AlaAla}-X).

In all cases, including JRK-{AlaAla}-X, which exhibited a limited solubility and a non-helical CD spectrum in the uncross-linked form, the resulting peptides were water-soluble and the secondary structures could be manipulated with light. Figure 3AGo (solid line) shows the CD spectrum for dark-adapted JRK-{AlaAla}-X with the azobenzene moiety in the trans conformation (Figure 3BGo). This spectrum is typical of largely disordered structure with a small degree of helicity (Johnson, 1990Go). Upon irradiation with 370 nm light, 72% of the cis conformation is obtained based on the observed UV spectrum (Fig. 3BGo, dotted line). The CD spectrum obtained after irradiation (Figure 3AGo, dotted line), shows that the intensity of the CD band at 222 nm has increased, the minimum at 200 nm has shifted to 207 nm and a strong maximum at 190 nm has appeared. This spectrum is characteristic of an {alpha}-helix (Greenfield and Fasman, 1969Go; Brahms and Brahms, 1980Go; Johnson, 1990Go; Reed and Reed, 1997Go). The CD spectrum expected for the 100% cis form of the peptide can be calculated simply by correcting the 72% cis spectrum for the presence of 28% trans; the resulting spectrum is shown in Figure 3AGo (dashed-dotted line).



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Fig. 3. Effects of photo-irradiation on JRK-{AlaAla}-X. (A) CD spectra of dark-adapted (trans) JRK-{AlaAla}-X (solid line) [45 µM, 5 mM sodium phosphate buffer (pH 7), 10 ± 1°C] and irradiated JRK-{AlaAla}-X (dotted line) [370 nm light, 3 min, 70 W]. Calculated CD spectrum for 100% cis (dashed-dotted line). (B) UV spectra of JRK-{AlaAla}-X, dark-adapted (trans) (solid line) and irradiated (dotted line). The spectrum of the 100% cis form of the cross-linker is shown as a dashed-dotted line.

 
Calculated values for the helix contents of the trans and cis forms of JRK-{ValAib}-X, JRK-{IleAib}-X, JRK-{AlaAla}-X and JRK-{hCys}{ValAib}-X are collected in Table IIGo. Helix contents of the peptides were calculated using the simple assumption that 100% helix gives a {theta}222 value of (–40 000) [(n 4)/n], where n is the number of residues (Kallenbach and Spek, 1998Go).

In all cross-linked peptides, the helix–coil structural transition observed by CD upon photo-isomerization was fully reversible with a half-life of ~0.5 h at room temperature. Also, all cross-linked peptides exhibited an isodichroic point at 203 nm, which suggests the existence of a simple two-state equilibrium between helical and disordered forms of the peptides (Rohl and Baldwin, 1998Go). As with the original system (Kumita et al., 2000Go), we found no evidence of self-association in any of the cross-linked peptide variants.

Molecular modelling of the cross-linked peptide variants

In the absence of a molecular mechanics parameterization for the azobenzene cross-linker or a combined MM/QM method, our original modelling approach treated the azobenzene group as rigid using heavy restraint terms (Kumita et al., 2000Go). Here, a combined molecular mechanics and ab initio quantum mechanics (MM/QM) approach (Svensson et al., 1996Go) was employed that allowed a more realistic treatment of the cross-linker.

Models were produced for the cis and trans states of each peptide as described in the Materials and methods section. Models for the cis and trans states of JRK-{ValAib}-X are shown in Figure 4Go. For each optimized model the extent of distortion from an ideal {alpha}-helical geometry was assessed by classifying the strength of all helical hydrogen bonds using the DSSP package (Kabsch and Sander, 1983Go). Results of the procedure are listed in Table IIIGo.



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Fig. 4. Energy optimized molecular models of JRK-{ValAib}-X. Models with the cross-linker in the cis form (a) and the trans form (b). Space filling representations of the cross-linker and Cys side chains, from Cb, are coloured violet. In cyan are the extra atoms of the amino acid side chains for Val and Aib compared with Ala and Ala. The peptide backbone is shown as a tube with colours assigned in accordance with the strength of the {alpha}-helical hydrogen bonds made on a residue by residue basis: blue = strong, green = intermediate, red = weak. A thick black line shows the deviation from planarity of the trans isomer required for it to accommodate an {alpha}-helical peptide backbone conformation. Figure prepared using VMD (Humphrey et al., 1996Go) and Raster3D (Merritt and Bacon, 1997Go).

 

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Table III. DSSP analysis of helix distortion in cross-linked peptides
 
The rationale behind the modelling procedure is that it quickly allows an assessment of how far each sequence/cross-linker conformation forces the peptide to move from an ideal {alpha}-helical conformation. It is expected that when a number of helical hydrogen bonds are weakened, the helical conformation would be disfavoured.

For each of the cross-linked peptide sequence variants, the trans form of the azobenzene cross-linker led to a high degree of helix destabilization as evidenced by a large number of weakened hydrogen bonds (Table IIIGo). In comparison, in all cases the cis cross-linker conformation was much more compatible with helicity (Table IIIGo). Comparing different sequences it can be seen that altering the underlying residues from small (JRK-{AlaAla}-X) to large (JRK-{ValAib}-X or JRK-{IleAib}-X) marginally increases the disruption of helicity for the trans conformation, producing three rather than two ‘weak’ helical hydrogen bonds.

Altering the length of the cross-linker by replacing cysteine residues with the longer homocysteine has a larger impact on calculated helix stability. For the trans conformation of the cross-linker, the hCys substitution significantly reduces the disruption of helicity (producing no ‘weak’ hydrogen bonds compared with three for the original peptide JRK-{ValAib}-X). For the cis conformation, the small amount of helix destabilization seen with the original peptide is relieved.

Effects of a non-cross-linking azobenzene modifier on JRK-{ValAib}

To test whether the photo-control of helix content observed with JRK-{ValAib}-X (and by extension with its variants) requires an intramolecular cross-link, each Cys of JRK-{ValAib} was reacted with the non-linking azobenzene reagent 2 (Figure 1BGo). CD spectra of the resulting peptide (JRK-{ValAib}-Mod) are shown in Figure 5Go. Mean residue ellipticities and calculated helix contents are given in Table IIGo. Dark-adapted JRK-{ValAib}-Mod, where the azobenzene groups are in the trans form, appeared about 60% helical (Figure 5AGo, solid line). Exposure to 340 nm light caused photo-isomerization to the cis form, as evidenced by changes in the UV spectra (Figure 5BGo), but there was little change in the degree of helicity (Figure 5AGo, dotted line).



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Fig. 5. Effects of photo-irradiation on JRK-{ValAib}-Mod peptide. (A) CD spectra of dark-adapted JRK-{ValAib}-Mod (solid line [39 µM, 5 mM sodium phosphate buffer (pH 7), 10°C] and irradiated JRK-{ValAib}-Mod (dotted line) [340 nm light, 3 min, 70 W]. (B) UV spectra of JRK-{ValAib}-Mod dark-adapted (solid line) and irradiated (dotted line).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptide sequence variants: uncross-linked

A number of relatively conservative sequence variants of JRK-{ValAib}-X were designed to test the importance of steric interactions between the cross-linker and underlying residues for the observed photo-control of helix content. The conformational behaviour of these peptides was first examined in uncross-linked form.

The increased helicity displayed by JRK-{IleAib} and JRK-{hCys}{ValAib} in the uncross-linked form as compared with JRK-{ValAib}, (Table IGo) may be attributed to the altered residue(s) having higher helical propensities than the original residues (Chou and Fasman, 1974Go; Williams et al., 1987Go; Creamer and Rose, 1994Go; Luque et al., 1996Go). The predictions of helix propensity using the AGADIR method (Munoz and Serrano, 1994Go) are necessarily limited by the necessity for substituting standard types for non-coded amino acids. However, given this limitation, it would be expected that substitution of Ile for Val would lead to an increase in helicity as found (Table IGo). In JRK-{hCys}{ValAib}, the two cysteine residues were replaced with two homocysteine residues, which increased the length of the side chains. Although the helical propensity of homocysteine has, to our knowledge, not been reported, the side chain structure is similar to methionine (minus the terminal methyl group). Various studies of helix preferences for individual amino acids agree that methionine has a higher helix propensity than cysteine (Chou and Fasman, 1974Go; Williams et al., 1987Go; O’Neil and DeGrado, 1990Go; Koehl and Levitt, 1999Go). This is borne out by AGADIR prediction (substituting Met for hCys), where a marked increase in helicity would be expected (Table IGo). The observed increase in helicity of JRK-{hCys}{ValAib} is thus probably due to homocysteine having a higher helix propensity than cysteine.

The peptide JRK-{AlaAla}, in which the (i + 3), (i + 4) residues were replaced with alanine, showed qualitatively different behaviour. The CD spectrum (Figure 2Go), with a minimum near 220 nm, suggested the presence of a ß-sheet conformation (Greenfield and Fasman, 1969Go; Brahms and Brahms, 1980Go; Johnson, 1990Go; Reed and Reed, 1997Go). Extended ß-sheet formation might also explain the limited solubility observed with the peptide. The work of Cerpa et al. examining the behaviour of a related peptide (Cerpa et al., 1996Go) provides a means for understanding ß-sheet formation in this case. Figure 2Go shows the JRK-{AlaAla} peptide modelled as an extended antiparallel ß-sheet. It will be noted that all the hydrophilic residues point in the same direction. If a number of such strands came together it would be possible to form an extended ‘ß-tape’ (Aggeli et al., 1997Go) with all main chain hydrogen bonds satisfied. One side of the tape would be hydrophilic with the arginine and glutamic acid residues forming networks of salt bridges. The other side would be flat and hydrophobic, giving the possibility of two such tapes associating with the hydrophobic faces contacting. It is interesting that the small variation in sequence between JRK-{AlaAla} and JRK-{ValAib} led to such alteration in behaviour; this type of ß-sheet disruption has been reported for model peptides in organic solvents (Moretto et al., 1989Go) and appears to be driven by the inability of Aib to adopt a ß-conformation (Marshall et al., 1990Go).

Importance of an intramolecular cross-link

The peptide JRK-{ValAib}-Mod, in which both Cys residues are each modified with an azobenzene moiety, showed no substantial change in secondary structure upon photo-isomerization. Thus, an intramolecular cross-link appears critical for photo-control of helix content in these peptides. Interestingly, JRK-{ValAib}-Mod showed a relatively high helix content (Table IIGo) compared with the other uncross-linked peptides (Table IGo). A helical structure might be stabilized by aromatic {pi}{pi} interactions between the two azobenzene side chains (Hunter and Sanders, 1990Go; Shoemaker et al., 1990Go). The azobenzene side chains are also flexible enough to interact with the peptide backbone and may stabilize the helical structure by affecting solvation of the backbone (Bodkin and Goodfellow, 1995Go). Whatever the mechanism(s) of helix stabilization in this case, however, comparable stabilizing factors must exist in both cis and trans states so that there is little net effect of photo-isomerization on secondary structure.

Peptide sequence variants: cross-linked

The behaviour of the cross-linked peptides contrasts markedly with that of the uncross-linked forms. In the dark, all the cross-linked peptides have a CD spectrum characteristic of a random coil with a small degree of helicity. Upon photo-isomerization there is a large increase in helicity in all cases. Helix destabilization by the trans form of the cross-linker therefore does not appear to require unfavourable steric interactions with underlying residues. While the helix contents observed for the trans forms of JRK-{AlaAla}-X and JRK-{hCys}{ValAib}-X are somewhat higher than for JRK-{ValAib}-X and JRK-{IleAIb}-X (Table IIGo), consistent with a minor role for steric interactions, these peptides are still substantially less helical than their corresponding cis forms. The somewhat lower helicity observed for the cis form of JRK-{hCys}{ValAib}-X is perhaps due to the longer cross-linked bridge being less conformationally restrictive and so providing less entropic stabilization of the helical state.

Not only does sequence variation not substantially affect photo-control, but also the cross-linker appears to override the individual intrinsic helical propensities of the amino acids used, in both trans and cis conformations. The effect of the cross-linker on the JRK-{AlaAla} peptide is particularly dramatic: cross-linking prevents ß-sheet formation.

Treating the cross-linker as a rigid unit in the original modelling exercise appears to have been the source of the mistaken hypothesis that bulky underlying residues would sterically interact with the trans conformation of the cross-linker producing a large increase in helix destabilization for this form. Allowing the cross-linker to relax using a combined MM/QM energy representation gives results that are consistent with experiment. The optimized models for JRK-{ValAib}-X produced using this improved procedure are shown in Figure 4Go. The trans form of the cross-linker can be seen to distort slightly to avoid steric clashes with the bulky residues. In comparison, the cis conformation of the cross-linker forces the large sulfur atoms of the cysteine close to the bulky underlying residues producing a small degree of (undesired) helix destabilization for this form (Figure 4bGo).

The hypothesis, although in error, had one serendipitous effect. To increase the bulk of the underlying residues an Aib residue was introduced into the original peptide sequence. This meant that the peptide was prevented from forming an unforeseen ß-sheet aggregate, considerably simplifying the protocol needed for cross-linking and the initial analysis.

Conclusion

For the present system, it appears that an intramolecular azobenzene cross-linker between residues i and i + 7 is sufficient to permit photo-control of helix content. Computational and experimental results agree that steric interactions between residues i + 3, i + 4 and the cross-linker are not critical for the photo-switching behaviour. The non-coded residue Aib is thus not required at position i + 4, which means this cross-linking strategy may be used in larger protein systems (in which synthesis by solid-phase methods is not practical). Interestingly, the cross-linker introduced photo-switching between unfolded and helical states in a peptide that existed as a ß-sheet in its native form (JRK-{AlaAla}), illustrating that this reagent is not limited to sequences containing a partial degree of helicity in their native states.


    Notes
 
3 To whom correspondence should be addressed. E-mail: awoolley{at}chem.utoronto.ca Back


    Acknowledgments
 
We acknowledge NSERC (Canada) (G.A.W.), the Volkswagen Stiftung (Germany) (G.A.W.) and the UK Medical Research Council [grant G.4600017 (O.S.S.)] for financial support. J.R.K. was supported by a Dr Dina Gordon Malkin/OGSST and an NSERC studentship. D.G.F. was supported by a UK Medical Research Council Bioinformatics studentship.


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Received December 1, 2001; revised March 8, 2002; accepted March 20, 2002.





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