K3 Heterodimeric Coiled Coil - ACS Publications

Oct 20, 2008 - Citation data is made available by participants in Crossref's Cited-by Linking service. For a more comprehensive list of citations to t...
10 downloads 0 Views 1MB Size
Biomacromolecules 2008, 9, 3173–3180

3173

pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil Bojana Apostolovic and Harm-Anton Klok* E´cole Polytechnique Fe´de´rale de Lausanne (EPFL), Institut des Mate´riaux, Laboratoire des Polyme`res Baˆtiment MXD, Station 12, 1015 Lausanne, Switzerland Received July 7, 2008; Revised Manuscript Received September 3, 2008

This manuscript reports on the self-assembly properties of two complementary peptide sequences, E3 and K3, which are derived from the known IAAL E3/K3 heterodimeric coiled coil motif. Circular dichroism spectroscopy, analytical ultracentrifugation, and fluorescence resonance energy transfer experiments indicated that a stoichiometric mixture of these two peptides forms a stable heterodimeric coiled coil at pH 7. At pH 5, in contrast, the E3/K3 heterodimeric coiled coil is unstable and unfolds to generate E3 homotrimers that coexist with K3 unimers and a small fraction of K3 homodimers. This pH-induced unfolding transition was unprecedented for this coiled coil motif but is of interest as it occurs within a physiologically relevant pH range, as it is encountered, for example, during cellular uptake via the endosomal pathway. This feature, in combination with the relatively short length of the E3 and K3 peptides and the high stability of the E3/K3 coiled coil at pH 7 makes this folding motif very attractive for the development of noncovalent polymer therapeutics and self-assembled biohybrid hydrogels.

Introduction The coiled coil is one of the most abundant protein folding motifs in Nature and consists of two to five R-helices that are wrapped around each other in a superhelical fashion.1-3 Coiled coil formation can occur between identical peptide sequences (homooligomerization) as well as between different peptide sequences (heterooligomerization). The coiled coil domains of many proteins are of crucial importance to their function and play an important role e.g. in the regulation of DNA transcription and viral fusion.4-7 With the current understanding of the structural features that determine the folding behavior of coiled coil peptides it is now possible to design peptide sequences with reasonable control over aggregation number, orientation, stability and specificity (i.e., homo- versus heterooligomerization).8-11 The ability to design sequences with predictable folding properties makes coiled coil peptides attractive building blocks for the development of novel self-assembled biomaterials.8,9,11,12 Among others, coiled coil peptides have attracted interest as structure-directing auxiliaries that mediate the self-assembly of peptides/proteins or peptide/protein-polymer conjugates into hydrogel networks or micellar aggregates. Because many coiled coils are sensitive to changes in temperature or pH, these hydrogels and micelles can be dissociated under the influence of appropriate environmental stimuli, which is an attractive feature for many biomedical applications. So far, most of the hydrogels and micellar assemblies that have been reported are based on peptides/proteins or peptide/protein-polymer conjugates containing homooligomeric coiled coil sequences.13-19 The use of homooligomeric coiled coil motifs, however, has several drawbacks, which include the possible formation of cycles and/or loop-type defects that may adversely influence the mechanical properties of the hydrogels. Furthermore, the use of homooligomeric coiled coil motifs prevents the design of graft-type constructs in which multiple biologically active molecules are attached to a synthetic polymeric carrier via a coiled coil linker. Both of these problems, however, can be overcome by using heterooligomeric coiled coil motifs with a * To whom correspondence should be addressed. E-mail: harm-anton.klok@ epfl.ch.

high binding specificity. Ideally, these heterooligomeric coiled coils should undergo reversible folding/unfolding transitions under (near) physiological conditions to allow access to stimulisensitive (bio)materials. In an earlier report, we have described the use of a heterodimeric coiled coil as a structure-directing auxiliary to mediate the formation of nanosized peptide-poly(ethylene glycol) micellar assemblies.20 This coiled coil motif, however, resulted in the formation of a mixture of dimeric and tetrameric assemblies that were insensitive to changes in pH. As a result, this motif lacked the specificity and sensitivity that would be desired for many biomaterials applications. Kopecˇek et al. have used a heterodimeric coiled coil motif to prepare hybrid hydrogels.21 This coiled coil motif consisted of two 39 amino acid long peptide sequences with opposite charges. Circular dichroism (CD) spectroscopy and analytical ultracentrifugation (AUC) experiments revealed the formation of stable heterodimeric coiled coils at neutral pH. Changing the pH to more acidic (pH 5) or basic (pH 9) conditions resulted in unfolding of the heterodimeric structures and in the formation of homodimeric coiled coils of one strand, which coexisted with unordered unimers of the other strand. Heterodimer formation between two complementary coiled coil peptides has also been explored by Harden et al. to promote hydrogel formation of ABC type artificial protein triblock copolymers.22 Our interest in heterodimeric coiled coils is due to their possible use as noncovalent linkers to prepare side chain functionalized polymer-drug conjugates. When appropriately designed peptide motifs are used, the drop in pH from pH ∼ 7 to pH ∼ 5 that would accompany the cellular uptake of such noncovalent polymer-drug conjugates via the endosomal pathway may be used to trigger intracellular drug release. Heterodimeric coiled coils have been proposed as binding motifs to allow pretargeted delivery of, for example, radionuclides.8,11,23 While pretargeted delivery and imaging takes advantage of the high heterospecificity and stability of appropriately designed heterodimeric coiled coils, no efforts have been reported so far to explore the possible pH-sensitivity of heterodimeric coiled coils to trigger intracellular drug release. This latter application would require a heterodimeric coiled coil that is characterized

10.1021/bm800746e CCC: $40.75  2008 American Chemical Society Published on Web 10/21/2008

3174

Biomacromolecules, Vol. 9, No. 11, 2008

by a very high stability and heterospecificity at pH 7 but which unfolds around pH 5. Although a number of pH-sensitive heterodimeric coiled coils has been reported, most of these are more stable under acidic conditions (pH 2-5) than at pH 7,24-30 which is opposite to what would be needed to trigger intracellular drug release. Very elegant heterooligomeric coiled coil motifs that can be switched in the desired pH range have been described by Schnarr and Kennan31 and Raydnov et al.32 The coiled coil motifs used by these groups, however, are not simple heterodimers, but more complex motifs, which may be attractive to develop pH-sensitive hydrogels, but which are less suited as noncovalent linkers to prepare noncovalent polymer-drug conjugates. A potentially very interesting heterodimeric coiled coil motif that may possess the desired pH-sensitivity is the IAAL E3/K3 heterodimeric coiled coil, which has been developed by Litowski and Hodges.33 One of the attractive features of the IAAL E3/ K3 coiled coil is that it consists of two relatively short peptide strands, composed of only 21 amino acids each. This is much shorter than e.g. the 39 amino acid long peptide sequences used by Kopecˇek, which facilitates synthesis and functionalization. The IAAL E3/K3 coiled coil consists of two oppositely charged peptide strands, which contain glutamic acid, respectively, lysine residues at positions “e” and “g” of the heptad repeat. The presence of the oppositely charged lysine and glutamic acid residues not only provides additional electrostatic interactions that promote the formation of stable heterodimers at neutral pH, but also potentially offers the possibility to manipulate the stability of the IAAL E3/K3 heterodimeric coiled coil via pH. So far, the pH sensitivity of the IAAL E3/K3 coiled coil has not been studied. In this report, we present the results from an extensive study on the pH sensitivity of the IAAL E3/K3 coiled coil. The results presented in this manuscript demonstrate that the IAAL E3/K3 heterodimeric coiled coil undergoes an interesting structural transition upon decreasing the pH from 7 to 5, which makes this peptide motif an attractive candidate for the synthesis of peptide/protein (hybrid) materials with interesting properties for a range of biomedical applications.

Experimental Section Materials. Fmoc-protected amino acids and Rink Amide AM resin (200-400 mesh, loading 0.71 mmol/g) were purchased from Novabiochem (brand of Merck Biosciences AG, Switzerland). HCTU (2(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium) and HOBt (N-hydroxybenzotriazole) were purchased from IRIS Biotech GmbH (Marktredwitz, Germany). NMP (N-methyl-2-pyrrolidone) was received from Schweizer Hall (Basel, Switzerland) and freshly distilled over calcium hydride and stored over 4 Å molecular sieves prior to use. 4-Methylmorpholine (NMM), acetic anhydride, piperidine, 1,8diazabicyclo[5.4.0]undec-7-ene (DBU), trifluoroacetic acid (TFA), acetonitrile (ACN), and triisopropylsilane (TIS) were used as received from Fluka Chemie GmbH (Buchs, Switzerland). Diethyl ether was purchased from Reactolab (Servion, Switzerland). Peptide Synthesis. Peptides were synthesized on a Chemspeed PSW 1100 peptide synthesizer using Fmoc chemistry and a Rink Amide AM resin support (0.1 mmol scale). Fmoc groups were removed using 1.5 mL of a 20% (v/v) solution of piperidine in NMP and amino acid coupling was carried out following a double coupling strategy with a 3-fold excess of Fmoc-amino acids (0.5 M solution in NMP). Coupling was achieved using a 1/0.5/1/2 ratio of amino acid/HOBt/HCTU/NMM in NMP. The deprotection and coupling times were 5 and 30 min, respectively. Three 1.5 mL NMP washes were performed between deprotection and coupling steps. After completion of the desired peptide sequence, the N-terminal amino acid was acetylated by treating the resin-bound peptide with 2 mL of an NMP solution containing 20%

Apostolovic and Klok (v/v) acetic anhydride and 15 mM of HOBt for 30 min. After that, the peptide-loaded resin was treated with a 95/2.5/2.5 (v/v) mixture of TFA/ TIS/water for 3 h, which simultaneously cleaved the peptide from the resin and removed all side-chain protective groups. The cleaved peptides were subsequently isolated by precipitation in cold diethyl ether followed by centrifugation. The precipitated peptides were resuspended in fresh diethyl ether and centrifuged (4 times). Finally, the peptides were dissolved in MilliQ water and lyophilized. Peptide Purification. After lyophilization, the peptides were dissolved in MilliQ water containing 0.1% TFA (v/v) and purified by reversed phase high performance liquid chromatography (RP-HPLC) using a Waters 600 automated gradient controller pump module, a Waters Prep Degasser, a Waters 2487 dual λ absorbance detector and a Waters Fraction Collector III. The preparative column, Atlantis dC18 OBDtm 5 µm, 30 × 150 mm column (Waters), was operated at a flow rate of 20 mL/min. Peptide purification was achieved using two mobile phases: mobile phase A consisted of a 0.1% solution of TFA in MilliQ water and mobile phase B consisted of a 0.1% solution of TFA in acetonitrile. The E3 peptides were purified using a linear AB gradient from 40% (v/v) to 55% (v/v) B over period of 25 min. For the K3 peptides a linear gradient from 27% (v/v) to 42% (v/v) B over 25 min was used. Results were processed with MassLynx V4.0 software (Waters). After purification, residual traces of TFA were removed as described elsewhere.34 Peptide purity was checked by using a Diphenyl analytical column 5 µm, 4.6 × 250 mm (Vydac) on the Waters instrument described above. Elution was achieved using a flow rate of 1 mL/min and a linear gradient from 5 (v/v) to 95% (v/v) of B over a period of 25 min. All peptides are g95% pure (Supporting Information). Mass Spectrometry. Mass spectrometry was performed on a single quadrupole mass spectrometer Finnigan SSQ 710C (Finnigan-MAT, Bremen, Germany) equipped with an electrospray (ES) ionization interface. Data were acquired using the ICIS software running on a Digital Unix workstation. Circular Dichroism Spectroscopy. Circular dichroism (CD) spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Jasco PTC-348WI temperature controller. Experiments were carried out using a 0.1 cm path length quartz cuvette. Spectra were recorded between 190 and 250 nm at a resolution of 1 nm with a time response of 1s. Each spectrum represents an average of 10 consecutive scans subtracted from the background. Mean residue molar ellipticity [Θ] in deg · cm2 · dmol-1 was calculated as follows

[ MRW 10lc ]

[Θ] ) [Θ]m

(1)

where [Θ]m is the ellipticity in millidegrees; MRW is the mean residue molecular weight of the peptide (MW/number of chromophores, i.e., amide bonds); l is the optical path length of the cuvette (cm) and c is the peptide concentration in 10 mM phosphate buffer (g/dm3). Peptide concentrations were determined using UV-vis spectroscopy, taking advantage of the UV-vis absorbance of tyrosine (280 ) 1480 M-1 · cm-1),35 EDANS-glutamic acid (339 ) 5900 M-1 · cm-1)36 or Dabcyl-lysine (453 ) 32000 M-1 · cm-1)37 residues present in the peptides. R-Helix contents were calculated from the observed mean residue molar ellipticity at 222 nm ([Θ]222) using the following equation

R-helix content )

[Θ]222 [Θ]222,th

(2)

where [Θ]222,th is the predicted molar ellipticity corresponding to 100% helix as calculated by38

(

[Θ]222,th ) -40 · 103 1 -

4.6 n

)

(3)

where n is the number of amino acid residues in the peptide. Thermal Unfolding Experiments. Temperature-dependent CD experiments were carried out to determine the unfolding enthalpy of the heterodimeric coiled coil at the transition midpoint Tm, which is the

E3/K3 Heterodimeric Coiled Coil

Biomacromolecules, Vol. 9, No. 11, 2008

temperature at which the fraction of monomeric peptide is 0.5. These experiments were carried out using an equimolar mixture of the E3 and K3 peptides at a total peptide concentration of 80 µM in 10 mM phosphate buffer at pH 7. Samples were subjected to two heating cycles; first, the sample was heated from 5 to 95 °C at a heating rate of 1 °C/min, then cooled down to 5 °C, allowed to equilibrate for 30 min at 5 °C and subsequently reheated from 5 to 95 °C at a heating rate of 1 °C/min. Each 5 °C, the sample was scanned over a wavelength range of 190 to 250 nm. The resulting thermal unfolding curves were subsequently analyzed using a two-state conformation transition model as described in reference.39 Determination of the Binding Constants. The binding constant of the E3/K3 heterodimeric coiled coil was determined by measuring the mean residue molar ellipticity at 222 nm of a series of solutions (10 mM phosphate buffer at pH 7) with a total peptide concentration of 80 µM but with varying ratios/mol fractions of the E3 and K3 peptides. Assuming a 1:1 binding stoichiometry and that [Θ]222 increases linearly with the fraction of coiled coil heterodimers in solution, the experimental results were analyzed with following equations:

∆Θmax ∆Θ ) ∆[AB] ∆[AB]max K) [Θ] ) [Θ]0 +

(4)

[AB] [AB] ) [A][B] [A0 - AB][B0 - AB]

[

(5)

(

1 + K([A]0 + [B]0) 2K

)

2

]

- [A]0[B]0 (6)

where ∆[Θ] ) [Θ] - [Θ]0; [Θ] is the measured mean residue molar ellipticity at 222 nm of the different peptide mixtures; [Θ]0 is the mean residue molar ellipticity at 222 nm for a 80 µM solution of E3; [A], [B], and [AB] are the equilibrium concentrations of E3, K3, and heterodimer E3/K3 respectively; [A]0 and [B]0 are loading concentrations of E3 and K3, respectively; ∆[Θ]max ) [Θ]max - [Θ]0; [Θ]max is the maximum absolute value of the mean residue molar ellipticity at 222 nm for the equimolar mixture of peptides when maximal concentration of the heterodimeric complex is formed ([AB]max). The binding constant K was obtained by nonlinear least-squares fitting of the experimental data with eq 6 using Matlab. Analytical Ultracentrifugation. Sedimentation equilibrium experiments were conducted on a Beckman XL-I centrifuge (Beckman, Palo Alto, CA) equipped with scanning UV-vis and Rayleigh interference optics using a An-60 Ti rotor and six channel cells with sapphire windows. Runs were carried out on samples with different peptide concentrations (40-400 µM) in 10 mM phosphate buffer (pH 7 and 5) at 25 °C with a rotor speed of 48000 rpm. Samples were dialysed for 24 h against 10 mM phosphate buffer prior to runs. Peptide concentrations were determined by UV-vis spectroscopy using known molar extinction coefficients, as described above. The partial specific volumes of the peptides40,41 as well as solvent densities42 were calculated as described in the literature. Equilibrium was typically established after ∼30 h when two consecutive scans taken 3 h apart could be perfectly superimposed. In equilibrium, the concentration distribution is exponential and the measured absorbance is a function of the radial position according to the following equation43

a(r) )

∑ n

[

cn,0εnd exp

compartments. First, data were fit using Sedpath44 by implementing the single species of interacting system model. This model gives an average molecular weight of the species present in the equilibrium. In the case of oligomerization, that is, when the calculated molecular weight is higher than that of the individual peptides, the data was fit using other proposed models by Sedphat. The final model was chosen according to the smallest value of root-mean-square deviation (rmsd) and where the distribution of error was random around zero mean deviation. The final model quantitatively describes the equilibrium present in the system, that is, existing species in the solution and gives the association constant for interactive peptides. Fluorescence Resonance Energy Transfer (FRET). FRET experiments were carried out using peptides labeled with 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS) as the donor and 4-((4(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) as the acceptor. Fluorescence spectra were recorded on a SPEX Fluorolog II spectrometer (Instruments S.A., Stanmore, U.K.) with a 1 cm path length quartz cuvette. Spectra were recorded from 350 to 750 nm with a resolution of 2 nm and an integration time of 1 s. The excitation wavelength was 339 nm. Fluorescence spectra of mixtures of appropriate EDANS and Dabcyl-labeled peptides were recorded at room temperature in 10 mM phosphate buffer at pH 7 and 5. The transfer efficiency (E) was calculated from the decrease in the fluorescence intensity at 504 nm of the EDANS donor using the following equation45

E ) 1 - (FDA ⁄ FD)

[Θ]max - [Θ]0 1 + K([A]0 + [B]0) [AB]max 2K

]

Mn(1 - jVnF)ω2 2 (r - r02) + δ 2RT

(7)

where cn,0 is the molar concentration of species n at a reference position r0; Mn, Vjn and n are the molar mass, the partial specific volume, and the molar extinction coefficient, respectively; d is the optical path length (1.2 cm); R is the universal gas constant; T is the absolute temperature, and δ is a baseline offset factor that compensates for differences in nonsedimenting absorbing solutes between sample and reference

3175

(8)

where FDA and FD represent the fluorescence intensity of the donor in the presence and absence of the acceptor, respectively. From the transfer efficiency E, the distance R between donor and acceptor can be calculated using45

E)

R06 R06 + R6

(9)

where R0 is the Fo¨rster radius (which is 33 Å for the EDANS/Dabcyl pair46). The binding constant (K) of the heterodimeric coiled coil was determined as for CD measurements using eq 6, assuming that the decrease in fluorescence intensity of the EDANS-labeled peptide upon addition of Dabcyl-labeled peptide is linearly proportional to heterodimer formation, because Dabcyl-labeled peptides have no emission. Isothermal Titration Calorimetry. ITC experiments were conducted at 25 °C on a Nano-ITC III (CSC5300) calorimeter (Calorimetry Science Corporation, Lindon, Utah). Peptide solutions were prepared by dissolving pure peptides in 10 mM phosphate buffer at pH 7. Prior to the experiment, 960 µL of a 40 µM solution of titrant was introduced in the sample vessel. The reference vessel was filled with 960 µL of a 10 mM phosphate buffer at pH 7. Then, a 400 µM solution of the titrate was introduced into the 250 µL rotating injection syringe. After baseline stabilization, injections of 5 µL of titrate solution with a relaxation time of 15 s were programmed with a delay of 300 s between each injection. Stirring rate in the reaction vessel was 200 rpm. The raw data was corrected for small heat changes upon injections after saturation was reached. These small heat changes were not directly related to association but were artifacts due to the heats of dilution, minor differences in temperature and composition of two mixing solutions. The binding stoichiometry (n), the association constant (K), and the molar enthalpy of folding (∆H) were obtained by nonlinear regression analysis to a 1:1 association model using ITC run version 1.1.0.28 and Bindworks version 3.1.7 software provided with the instrument.

Results and Discussion Peptide Design and Synthesis. The objective of the work described in this manuscript is to study the feasibility of the IAAL E3/K3 heterodimeric coiled coil33 as a pH-sensitive switch for the construction of “smart” self-assembled bioma-

3176

Biomacromolecules, Vol. 9, No. 11, 2008

Apostolovic and Klok

Table 1. Peptide Primary Structures and Molecular Weights peptide

primary structure

MWe (g/mol)

IAAL E3a IAAL K3a E3b,c,d K3b,c,d E3-ED K3-ED, E3-D

Ac-E(IAALEKE)2IAALEK-NH2 Ac-K(IAALKEK)2IAALKE-NH2 Ac-GYE(IAALEKE)2IAALEKG-NH2 Ac-GYK(IAALKEK)2IAALKEG-NH2 Ac-GE(IAALEKE)2IAALEKGE(EDANS)G-NH2 Ac-GE(EDANS)K(IAALKEK)2IAALKEG-NH2 Ac-GK(DABCYL)E(IAALEKE)2IAALEKG-NH2

2324 2321 2601 2598 2872 2812 2817

a Original sequence, as reported by Litowsky and Hodges.33 b,c,d IAAL E3 and IAAL K3 analogues used in this study. Peptides used for analytical ultracentrifugation experiments. Peptides used for FRET studies. e Peptide molecular weight (verified by ESI-MS). Ac and NH2 represent N-terminal acetyl, respectively, C-terminal amide end groups of the peptides.

terials. The choice of the IAAL E3/K3 motif was motivated by a number of factors. First of all, the IAAL E3/K3 coiled coil consists of two relatively short peptide chains, which facilitates synthesis and further functionalization. Second, despite its short length, the IAAL E3/K3 coiled coil is characterized by a high stability, which is illustrated by a very low dissociation constant (KD) of 7 × 10-8 M at pH 7. The heterodimeric nature and stability of the IAAL E3/K3 coiled coil at pH 7 are promoted by the presence of complementary charged Glu and Lys residues at positions “e” and “g” of the IAAL E3 and K3 strands, respectively. These charged residues, however, also potentially allow to manipulate the stability and folding/unfolding of the IAAL E3/K3 coiled coil, which is the main focus of the present work. The experiments described in this paper have been carried out with two analogues of the IAAL E3 and IAAL K3 peptides, which are referred to as E3 and K3, respectively, and which were extended with a Gly-Tyr diad at the N-terminus and an additional Gly residue at the C-terminus (Table 1). The Tyr residue was introduced to allow the determination of peptide concentrations via UV-vis spectroscopy and the presence of the additional Gly residues is expected to facilitate future Cand N-terminal modification of these peptides. Also listed in Table 1are two EDANS and one Dabcyl labeled peptide, which were designed and prepared for analytical ultracentrifugation and FRET studies. All peptides were prepared by standard Fmoc solid phase synthesis on a Rink amide resin, purified by preparative RPHPLC, and characterized by ESI mass spectrometry. Circular Dichroism Spectroscopy. Circular dichroism spectroscopy (CD) was used to study the secondary structure and folding behavior of the E3 and K3 peptides. At pH 7, the individual peptides show typical random-coil like spectra (Figure 1A). The CD spectrum of an equimolar mixture of the peptides at pH 7, however, shows the typical features of an R-helical secondary structure with two minima at 208 and 222 nm. This is a first indication that heterooligomeric assemblies composed of equimolar amounts of the E3 and K3 peptides are formed. The CD spectrum of the equimolar E3/K3 mixture is characterized by a very high helix content (∼97%). Furthermore, the ratio of the intensities at the minima in the CD spectrum of the equimolar E3/K3 mixture ([Θ]222/[Θ]208) is close to 1, which is a characteristic feature for coiled coil formation.47,48 CD spectroscopy was also used to determine the binding stoichiometry and binding constant of the E3/K3 complex. Figure 2 plots [Θ]222 as a function of the mole fraction of E3 for different mixtures of the peptides E3 and K3 at a constant total concentration of 80 µM. [Θ]222 reaches a minimum at a mole fraction of 0.5 of the E3 peptide, indicating a 1:1 binding stoichiometry. Fitting the data in Figure 2 yields a binding constant of K ) 6.85 × 107 M-1 (Table 2 and the Supporting Information). This binding constant is in reasonable agreement with the dissociation constant that was obtained for the IAAL

E3/K3 heterodimeric coiled coil by Litowski and Hodges using GdnHCl denaturation. From the binding constants, the concentrations and mole percentages of the free, i.e., unimeric and folded, i.e., present as heterodimers, peptides can be calculated, which are also listed in Table 2. Temperature-dependent CD experiments revealed a two-state helix-to-random coil transition with a transition midpoint temperature (Tm) of 70 °C and an unfolding enthalpy (∆Hm) of ∼130 KJ/mol (Figure 1B and the Supporting Information). Interestingly, the transition midpoint and unfolding enthalpy are comparable to that of the much longer (56 amino acid) coiled coil region of the bZIP transcription factor GCN4.49 The results from the temperature-dependent CD experiments were confirmed by isothermal titration calorimetry studies, which revealed a binding constant K of 2.76 × 107 M-1 (Table 2 and the Supporting Information). Furthermore, CD experiments carried out at pH 7 in different buffers and at different sample concentrations indicated that the E3/K3 coiled coil is relatively insensitive toward changes in ion strength (Figure 1C) and is also not significantly affected by dilution (Figure 1D). The data presented so far indicate that the E3 and K3 variants behave similar to the IAAL E3 and K3 peptides in that they are able to form stable heterodimeric coiled coils at pH 7. In a next series of CD experiments, the pH sensitivity of the E3/K3 coiled coil was investigated, which has not been studied before. Figure 3 compares the CD spectra of an equimolar mixture of the E3 and K3 peptides with that of the individual peptides at a total peptide concentration of 80 µM at pH 5. Whereas the CD spectrum of the K3 peptide is characteristic for a random coil, the CD spectra of the E3 peptide, and of the equimolar E3/K3 mixture show the typical features of an R-helical secondary structure. As the K3 peptide alone does not adopt an ordered secondary structure and because coiled coil forming peptides are unstable as unimers and tend to aggregate, the high helix contents in the CD spectra of the E3 peptide and of the E3/K3 mixture suggest the formation of E3 homooligomers at pH 5. The remainder of this manuscript will focus on elucidating the structural changes that accompany the change from pH 7 to 5 in a solution containing an equimolar mixture of E3 and K3. Analytical Ultracentrifugation. To reconfirm the binding stoichiometry of the E3/K3 coiled coil at pH 7 and, more importantly, to investigate the structural changes that occur upon decreasing the pH of a solution containing stoichiometric amounts of the E3 and K3 peptides from 7 to 5, sedimentation equilibrium experiments were carried out. The corresponding concentration profiles together with the residuals from the curve fit are provided in the Supporting Information. The numerical results from the sedimentation equilibrium experiments are summarized in Table 3. At pH 7, the molecular weight determined by AUC for the E3 peptide is in good agreement with the expected molecular weight, which indicates that this peptide exists as a unimer at pH 7. Sedimentation equilibrium experiments at pH 7 with the K3 peptide revealed a molecular weight that is slightly higher than the expected molecular weight, suggesting that the K3 peptide at pH 7 has a light tendency to aggregate. The experimentally determined molecular weight for a stoichiometric mixture of the E3 and K3 peptides at pH 7 is in good agreement with that of a dimer, which confirms the heterodimeric character of the E3/K3 coiled coil. Table 3 also indicates that the binding constant for the formation of the E3/K3 heterodimeric coiled coil is 4-6 orders of magnitude larger than that for homodimerization of the K3 peptides at pH 7. Consequently, heterodimer-

E3/K3 Heterodimeric Coiled Coil

Biomacromolecules, Vol. 9, No. 11, 2008

3177

Figure 1. (A) CD spectra of the peptides E3 (triangles), K3 (squares), and their equimolar mixture E3/K3 (black dots) measured in 10 mM phosphate buffer at pH 7. Peptide concentration of all samples was 80 µM. (B) Temperature-dependent CD spectra of an equimolar E3/K3 mixture dissolved in 10 mM phosphate buffer at pH 7 at a total peptide concentration of 80 µM, as recorded between 5 and 95 °C. The insert shows the temperature dependence of [Θ]222. (C) Comparison of CD spectra of an equimolar E3/K3 mixture (80 µM) in HEPES buffer, MilliQ water, and 10 and 150 mM phosphate buffer. (D) Comparison of the CD spectra of an equimolar mixture of the E3 and K3 peptides in 10 mM phosphate buffer at pH 7 at different total peptide concentrations. Table 2. Binding Constants and Equilibrium Concentrations of Unimeric Peptide and E3/K3 Heterodimers, as Obtained Using Different Experimental Techniques methoda b

literature CD ITC AUC FRET

K (M-1) 1.43 × 10 6.85 × 107 2.76 × 107 1010 4.57 × 107 7

[peptide]free (µM)

[heterodimer] (µM)

1.66 (4.15%) 0.76 (1.9%) 1.19 (2.97%) 0.03 (0.07%) 0.93 (2.3%)

38.34 (95.85%) 39.24 (98.1%) 38.81 (97.03%) 39.97 (99.93%) 39.07 (97.7%)

a Initial peptide concentration was the same in all experiments, [E3] ) [K3] ) 40 µM in 10 mM phosphate buffer at pH 7 and 25 °C. b Determined from Gdn HCl denaturation (see ref 33).

Figure 2. Mean residue molar ellipticity at 222 nm ([Θ]222) for mixtures of the E3 and K3 peptides as a function of the mole fraction of the E3 peptide. Experiments were carried out in 10 mM PB buffer at pH 7 at a total peptide concentration of 80 µM.

ization of E3 and K3 at pH 7 is not significantly influenced by homodimerization of K3. It is interesting to note that the binding constant for the formation of the E3/K3 heterodimer at pH 7 at a total peptide concentration of 80 µM as determined by AUC is approximately 3 orders of magnitude larger than the corresponding binding constants determined by CD or ITC. Sedimentation equilibrium analysis can provide relatively precise values for binding constants in the range of 103 to 108 M-1.43,50 Because CD and ITC revealed binding constants on the order of 107 - 108 M-1, which is close to the upper limit accessible

for AUC, we speculate that the observed discrepancy is due to the limited accuracy of AUC in characterizing these relatively strong interacting systems. To facilitate the interpretation of the sedimentation equilibrium results at pH 5, an additional variant of the E3 and K3 peptides was prepared, which contains an EDANS (5-((2aminoethyl)amino)naphthalene-1-sulfonic acid) modified glutamic acid residue at the penultimate C- or N-terminal position (Table 1). The EDANS label has a UV-vis absorbance at 339 nm, which does not overlap with the UV-absorbance of any other intrinsic peptide chromophore and thus allows to selectively monitor the oligomerization behavior of this labeled peptide in a mixture that also contains the complementary nonlabeled peptide. Sedimentation equilibrium analysis of the individual E3 and K3 peptides, both with and without EDANS label, at pH 5 indicated that the former peptide almost exclusively forms homotrimers, while the latter preferentially exists as a unimer (Table 3). CD experiments on E3-ED and its stoichiometric mixture with K3 at pH 7 and pH 5 (Supporting

3178

Biomacromolecules, Vol. 9, No. 11, 2008

Figure 3. CD spectra of peptides E3, K3, and their equimolar mixture (E3/K3) measured in 10 mM phosphate buffer at pH 5. Peptide concentration of all samples was 80 µM.

Information) also confirmed that the folding behavior of the labeled peptides was similar to that of the nonlabeled peptides. Minor, quantitative differences may be due to the presence of the labels. The results of the sedimentation equilibrium analysis on the individual peptides were confirmed by experiments that were carried out with 1: 1 mixtures of labeled and nonlabeled peptides using a scanning wavelength of 339 nm. The experiments carried out with the EDANS labeled E3 peptide yielded a molecular weight which is slightly below that expected for the E3-ED heterotrimer (8616 g/mol). This difference may be attributed to the presence of a small fraction of residual E3-ED/K3 heterodimers. Sedimentation equilibrium analysis of an equimolar mixture of E3 and the EDANS labeled K3 peptide afforded a molecular weight of ∼6000 g/mol. This is significantly larger than the 2812 g/mol of the K3-ED unimer, which would have been expected in the ideal case and also points toward the existence of residual, nondissociated E3/K3 heterodimer. Detailed, quantitative analysis of the composition of the peptide mixture at pH 5 is complicated by fact that the molecular weights of the E3/K3-ED heterodimer (5413 g/mol) and the K3-ED homodimer (5624 g/mol) are relatively similar. Based on the results of the AUC experiments on the individual peptide, however, a pH 5 solution probably contains E3 homotrimer that coexists with K3 unimers and dimers and as well as residual E3/K3 heterodimer. In summary, the results from the sedimentation equilibrium experiments unambiguously confirm the heterodimeric character of E3/K3 coiled coil at pH 7. The experiments carried out at pH 5 suggest that this drop in pH leads to a structural rearrangement and unfolding of the E3/K3 heterodimer. The experimental data suggest that at pH 5 a stoichiometric mixture of the E3 and K3 peptides mainly consists of E3 homotrimers that coexist with K3 unimers (and dimers) and residual E3/K3 heterodimer (Figure 4). Fluorescence Resonance Energy Transfer. The formation of the E3/K3 heterodimeric coiled coil and the pH induced unfolding was further studied using fluorescence resonance energy transfer (FRET) experiments. For this purpose, two derivatives of the E3 and K3 peptides were prepared. The penultimate N-terminal position of the K3 peptide was modified with a fluorescence donor EDANS (ED, 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), whereas the penultimate Nterminal amino acid of the E3 peptide was substituted with the 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl, D) ac-

Apostolovic and Klok

ceptor (Table 1). The donor and acceptor moieties were introduced at the N-terminus of both peptides because the E3/ K3 coiled coil consists of two parallel oriented strands,51 which would lead to fluorescence quenching upon formation of the coiled coil. Circular dichroism scans of (mixtures of) these labeled peptides were similar to the scans of the unmodified E3/K3 peptides that were presented in Figure 1A, which demonstrates that the folding properties of the peptides were not significantly affected by the presence of the dyes (Supporting Information). Figure 5 compares the fluorescence emission spectra of the individual EDANS and Dabcyl-labeled peptides at pH 5 and 7 with those of a 1:1 mixture of both peptides at the same pH values. The strong decrease in fluorescence intensity at pH 7 upon the addition of a stoichiometric amount of E3-D to a solution containing K3-ED is consistent with the formation of the heterodimeric coiled coil. From these spectral changes, a distance between donor and acceptor of 26 Å can be calculated. This distance is in good agreement with the estimated diameter of the heterodimeric coiled coil (D ∼ 3 nm).52 This result does not unambiguously reconfirm the parallel orientation of the E3/ K3 heterodimer. This is due to the fact that the EDANS and Dabcyl labeled peptides are only composed of 24 amino acids, which results in an estimated length of 35 Å of the coiled coil. Reducing the pH of a solution containing stoichiometric amounts of K3-ED and E3-D from pH 7 to 5 results in an increase in the fluorescence intensity. This observation is consistent with the unfolding of the E3/K3 heterodimer and the formation of E3 homotrimers that coexist with K3 unimers (and dimers) (Figure 4). The difference in fluorescence intensity at pH 5 between a solution containing a stoichiometric mixture of E3-D and K3-ED and a solution containing just K3-ED, however, indicates that only part of the E3/K3 heterodimer dissociates and that the solution at pH 5 still contains residual E3/K3 heterodimer. This observation is in agreement with the results from the AUC experiments (Vide supra). Figure 6 shows a series of fluorescence emission spectra of solutions containing 40 µM K3-ED and increasing concentrations of E3-D (0-60 µM). Comparison of the spectra reveals a continuous decrease in fluorescence intensity, which is in agreement with the formation of the E3/K3 dimer. With increasing concentration of E3-D, the spectra shown in Figure 6 also reveal a red shift of the emission maximum. As shown in the insert of Figure 6, the EDANS fluorescence intensity decreases linearly with increasing concentration of E3-D until a quencher concentration of about 40 µM, that is, a mixture containing stoichiometric amounts of the K3-ED and E3-D peptides. Assuming a two state model, the data in the insert in Figure 6 can be fitted to afford a binding constant for the formation of the K3/E3 heterodimer of ∼4.57 × 107 M-1 (Table 2 and Supporting Information), which is in good agreement with the binding constant reported in literature and the values determined using CD and ITC.

Conclusions The main objective of this manuscript was to study the possible pH-sensitivity of the IAAL E3/K3 heterodimeric coiled coil. The experiments that have been discussed were carried out with two variants of the IAAL E3 and IAAL K3 peptides, which were slightly modified at their N- and C-termini. The self-assembly of these two peptides, E3 and K3, and in particular the influence of pH was investigated using CD spectroscopy, analytical ultracentrifugation and FRET experiments. At pH 7, these experiments revealed that the E3 and K3 peptides, when

E3/K3 Heterodimeric Coiled Coil

Biomacromolecules, Vol. 9, No. 11, 2008

3179

Table 3. Numerical Results of AUC Experiments pH

λb (nm)

cc (µM)

MWd (AUC)

MWe (ESI)

E3

7

275

7

275

E3/K3a

7

275

E3

5

275

E3-ED

5

339

K3

5

275

K3-ED

5

339

E3-ED/K3a

5

339

E3/K3-EDa

5

339

2540 2876 2426 3202 3615 3436 4997 5436 5277 7081 7685 7952 9064 9647 9086 3357 3190 3734 3705 3861 3675 7488 7675 7371 6010 6003 5604

2601

K3

40 80 400 40 80 400 40 80 400 40 80 400 40 80 160 40 80 160 40 80 200 40 80 160 40 80 400

sample

2598 5199

Log10K1,2f

2872

2812

unimer (%)

dimer (%)

20 39 15 99.5 99.8 99.9

10.89 11.37 24.72 22.77 23.08 20.85

80 61 85 0.5 0.2 0.1 13 6 0 0 0 0 65 75 52 81 77 87

3.51 3.82 3.12 7.95 10 9.98

2601

2598

Log10K1,3g

4.02 3.45 3.76 3.94 3.76 2.97

trimer (%)

87 94 100 100 100 100 35 25 48 19 23 13

2872h 2812i

a Equimolar peptide mixture. b Scanned wavelength (nm). c Total peptide concentration (µM). d Calculated molecular weight determined using the single species of interacting system model. e Molecular weight of peptides obtained by ESI-MS. f K values calculated using heterodimer A + B ) AB model or monomer-dimer self-association model. g K values calculated using monomer-trimer self-association 3A ) A3 model. h Molecular weight of E3-ED. i Molecular weight of K3-ED.

Figure 4. Schematic presentation of the self-assembly of peptides E3 (blue) and K3 (green) at pH 7 and pH 5.

Figure 6. Fluorescence emission spectra of 10 mM phosphate buffer solutions at pH 7 containing 40 µM K3-ED and increasing concentrations of E3-D. The insert shows the fluorescence intensity at 504 nm as a function of the E3-D concentration.

Figure 5. Fluorescence emission spectra of E3-D, K3-ED, and their equimolar mixture in 10 mM phosphate buffer at pH 7 and 5.

mixed in stoichiometric quantities, form a stable heterodimeric coiled coil with a binding constant K of ∼7 × 107 M-1 (CD), similar to what is known for the IAAL E3/K3 heterodimer. At pH 5, in contrast, the E3/K3 coiled coil looses stability and

partially unfolds to generate E3 homotrimers that coexist with K3 unimers and a small fraction of K3 homodimers. This pHinduced unfolding transition was unprecedented for the E3/K3 heterodimeric coiled coil, but makes this folding motif very attractive for e.g. the development of noncovalent polymer therapeutics or self-assembled biohybrid hydrogels. The attractiveness of the E3/K3 coiled coil for such applications is due to (i) the high stability at pH 7 (K ∼107 M-1); (ii) the relatively short, 21 amino acid long sequences, which facilitate synthesis and functionalization; and (iii) the fact that unfolding can be induced by small changes in pH in a physiologically relevant pH range, which offers prospects, for example, for intracellular drug release.

3180

Biomacromolecules, Vol. 9, No. 11, 2008

Acknowledgment. Luc Benoist and Christian Peronnet from SETARAM Instruments (Lyon, France) are gratefully acknowledged for their assistance with the ITC experiments. We thank Dr. Christine Wandrey and Dr. Peter Schuck for many helpful discussions regarding sedimentation equilibrium experiments. We are grateful to Dr. Rudolf Hovius and Luigino Grasso for their help with the FRET experiments. This research is supported by the Swiss National Science Foundation and the NCCR Nanoscale Science. Supporting Information Available. Peptide characterization (HPLC and ESI mass spectra) and additional CD, AUC, and FRET results. This information is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Lupas, A. Trends Biochem. Sci. 1996, 21, 375–382. (2) Burkhard, P.; Stetefeld, J.; Strelkov, S. V. Trends Cell Biol. 2001, 11, 82–88. (3) Mason, J. M.; Arndt, K. M. ChemBioChem 2004, 5, 170–176. (4) Baxevanis, A. D.; Vinson, C. R. Curr. Opin. Genet. DeV. 1993, 3, 278–285. (5) Shaulian, E.; Karin, M. Nat. Cell Biol. 2002, 4, E131–E136. (6) Brown, N. L.; Stoyanov, J. V.; Kidd, S. P.; Hobman, J. L. FEMS Microbiol. ReV. 2003, 27, 145–163. (7) Eferl, R.; Wagner, E. F. Nat. ReV. Cancer 2003, 3, 859–868. (8) Hodges, R. S. Biochem. Cell Biol. 1996, 74, 133–54. (9) Woolfson, D. N.; Ryadnov, M. G. Curr. Opin. Chem. Biol. 2006, 10, 559–567. (10) Bromley, E. H. C.; Channon, K.; Moutevelis, E.; Woolfson, D. N. ACS Chem. Biol. 2008, 3, 38–50. (11) Yu, Y. B. AdV. Drug DeliVery ReV. 2002, 54, 1113–1129. (12) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2, 822–835. (13) Yang, J.; Xu, C.; Kopecˇkova´, P.; Kopecˇek, J. Macromol. Biosci. 2006, 6, 201–209. (14) Wang, C.; Stewart, R. J.; Kopecˇek, J. Nature 1999, 397, 417–420. (15) Pechar, M.; Kopecˇkova´, P.; Joss, L.; Kopecˇek, J. Macromol. Biosci. 2002, 2, 199–206. (16) Vandermeulen, G. W. M.; Tziatzios, C.; Klok, H. A. Macromolecules 2003, 36, 4107–4114. (17) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389–392. (18) Fischer, S. E.; Liu, X.; Mao, H.-Q.; Harden, J. L. Biomaterials 2007, 28, 3325–3337. (19) Wang, C.; Kopecˇek, J.; Stewart, R. J. Biomacromolecules 2001, 2, 912–920. (20) Vandermeulen, G. W. M.; Tziatzios, C.; Duncan, R.; Klok, H. A. Macromolecules 2005, 38, 761–769. (21) Yang, J.; Xu, C.; Wang, C.; Kopecˇek, J. Biomacromolecules 2006, 7, 1187–1195. (22) Mi, L.; Fischer, S.; Chung, B.; Sundelacruz, S.; Harden, J. L. Biomacromolecules 2006, 7, 38–47. (23) Moll, J. R.; Ruvinov, S. B.; Pastan, I.; Vinson, C. Protein Sci. 2001, 10, 649–655.

Apostolovic and Klok (24) Marti, D. N.; Jelesarov, I.; Bosshard, H. R. Biochemistry 2000, 39, 12804–12818. (25) Suzuki, K.; Yamada, T.; Tanaka, T. Biochemistry 1999, 38, 1751– 1756. (26) O’Shea, E. K.; Rutkowski, R.; Kim, P. S. Cell 1992, 68, 699–708. (27) Yu, Y.; Monera, O. D.; Hodges, R. S.; Privalov, P. L. Biophys. Chem. 1996, 59, 299–314. (28) Zhou, N. E.; Kay, C. M.; Hodges, R. S. Protein Eng. 1994, 7, 1365– 1372. (29) Kohn, W. D.; Kay, C. M.; Hodges, R. S. J. Mol. Biol. 1997, 267, 1039–1052. (30) Stevens, M. M.; Flynn, N. T.; Wang, C.; Tirrell, D. A.; Langer, R. AdV. Mater. 2004, 16, 915–918. (31) Schnarr, N. A.; Kennan, A. J. J. Am. Chem. Soc. 2003, 125, 6364– 6365. (32) Ryadnov, M. G.; Ceyhan, B.; Niemeyer, C. M.; Woolfson, D. N. J. Am. Chem. Soc. 2003, 125, 9388–9394. (33) Litowski, J. R.; Hodges, R. S. J. Biol. Chem. 2002, 277, 37272–37279. (34) Cornish, J.; Callon, K. E.; Lin, C. Q. X.; Xiao, C. L.; Mulvey, T. B.; Cooper, G. J. S.; Reid, I. R. Am. J. Physiol-Endocrinol. Metab. 1999, 277, E779–E783. (35) Mach, H.; Middaugh, C. R.; Lewis, R. V. Anal. Biochem. 1992, 200, 74–80. (36) http://probes.invitrogen.com/servlets/datatable?item)91&id)32085. (37) http://probes.invitrogen.com/handbook/tables/1407.html. (38) Gans, P. J.; Lyu, P. C.; Manning, M. C.; Woody, R. W.; Kallenbach, N. R. Biopolymers 1991, 31, 1605–1614. (39) Lavigne, P.; Crump, M. P.; Gagne´, S. M.; Hodges, R. S.; Kay, C. M.; Sykes, B. D. J. Mol. Biol. 1998, 281, 165–181. (40) Durchschlag, D. Specific volumes of biological macromolecules and some other molecules of biological interest. In Thermodynamic data for biochemistry and biotechnology; Hinz, H.-J., Ed.; Springer: Berlin, Germany, 1986; Chapter 3. (41) Durchschlag, H.; Zipper, P. Prog. Colloid Polym. Sci. 1994, 94, 20– 39. (42) Laue, T. M.; Shah, B. D.; Ridgeway, T. M.; Pelletier, S. L. Horton, J. C. Computed-aided interpretation of analytical sedimentation data for proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science; Harding, S. E., Rowe, A. J., Horton, J. C., Eds., Royal Society of Chemistry: Cambridge, England, 1992; Chapter 7. (43) Lebowitz, J.; Lewis, M. S.; Schuck, P. Protein Sci. 2002, 11, 2067– 2079. (44) http://www.analyticalultracentrifugation.com/sedphat/sedphat.htm. (45) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (46) http://probes.invitrogen.com/handbook/boxes/0422.html. (47) Zhou, N. E.; Kay, C. M.; Hodges, R. S. Biochemistry 1992, 31, 5739– 5746. (48) Zhou, N. E.; Kay, C. M.; Hodges, R. S. J. Biol. Chem. 1992, 267, 2664–2670. (49) Thompson, K. S.; Vinson, C. R.; Freire, E. Biochemistry 1993, 32, 5491–5496. (50) Hensley, P. Structure 1996, 4, 367–373. (51) Lindhout, D. A.; Litowski, J. R.; Mercier, P.; Hodges, R. S.; Sykes, B. D. Biopolymers 2004, 75, 367–375. (52) O’Shea, E. K.; Klemm, J. D.; Kim, P. S.; Alber, T. Science 1991, 254, 539–544.

BM800746E