Incorporation of Designed Extended Chromophores into Amphiphilic 4

NANO LETTERS

Incorporation of Designed Extended Chromophores into Amphiphilic 4-Helix Bundle Peptides for Nonlinear Optical Biomolecular Materials

2006 Vol. 6, No. 11 2387-2394

Ting Xu, Sophia P. Wu, Ivan Miloradovic, Michael J. Therien, and J. Kent Blasie* Department of Chemistry, UniVersity of PennsylVania, 231 South 34th Street, Philadelphia, PennsylVania 19104 Received September 5, 2006; Revised Manuscript Received September 26, 2006

ABSTRACT Multipigment ensembles that feature (porphinato)metal components and appropriate ethyne- and oligoyne-based chromophore-to-chromophore connectivity can manifest large optical polarizabilities and hyperpolarizabilities by design. Their vectorial orientation and local environment are controlled upon incorporation into designed amphiphilic 4-helix bundle peptides via axial histidyl ligation without disturbing the peptide’s helical secondary structure. The chromophore/peptide stoichiometry can be tuned by varying the peptide’s oligomeric state. The chromophore/ peptide complexes are thermally stable, making them ideal candidates for the fabrication of nonlinear optical biomolecular materials.

Introduction. Chromophores possessing extended π-electron systems can be designed with extraordinary electro-optical and nonlinear optical properties.1-4 Multipigment ensembles that feature (porphinato) metal components and appropriate ethyne- and oligoyne-based chromophore-to-chromophore connectivity can manifest substantial ground- and excitedstate interchromophore electronic interactions and a wide range of particularly impressive electro-optic properties.5-19 Such species can be tailored to exhibit substantial low-energy transition oscillator strengths, small potentiometric band gaps,11,16,18 and large polarizabilities and hyperpolarizabilities.8,9,11-14,19 Furthermore, these chromophoric elements can be utilized to design supermolecules that feature multiple coupled charged-transfer oscillators providing for extraordinary dynamic hyperpolarizabilities.9,19 To optimize the utility of these extended optical chromophores as a functional material, they must be oriented in an ensemble with uniform acentric order on a macroscopic scale, as in a thin film or fiber. Importantly, the chromophorechromophore interactions must also be controlled. Several methods have been employed to produce oriented ensembles of porphyrin-based chromophore systems.20-22 However, theoretical calculations suggest that the lack of control over the interactions between chromophores in these systems is most likely responsible for their inability to achieve predicted material performance.23,24 Achieving both of these requirements simultaneously in bulk materials thus remains extraordinarily challenging. * Corresponding author. E-mail: [email protected]. 10.1021/nl062091p CCC: $33.50 Published on Web 10/20/2006

© 2006 American Chemical Society

Incorporation of such extended chromophores into the interior of designed, amphiphilic 4-helix bundle peptides provides for both control of the local environment of the chromophore and its orientational order within an ensemble of peptide-chromophore complexes. Artificial proteins (or “maquette” peptides), based on R-helical bundle structural motifs, have been designed to incorporate biological cofactors, utilizing axial ligation coordination chemistry in the case of metallo-porphyrins.25-29 These “maquettes” retain a broad range of functionality imparted by the cofactors comparable to their natural counterparts, but within much simpler structures.30,31 Recent developments in de noVo computational protein design enable the design of peptides capable of binding abiological cofactors, such as the conjugated multichromophore ensembles noted above.32,33 These de noVodesigned proteins exploit axial ligation coordination chemistry as one tool to bind such abiological chromophore systems that contain a metalloporphyrin. The interior of the R-helical bundle scaffold can be designed to control the position, local environment, and orientation of the extended, conjugated chromophore within the peptide, and the exterior of the bundle can be used to control the peptide’s supramolecular assembly into an ordered ensemble of oriented peptide-chromophore complexes on a macroscopic lengthscale. Such engineering is an essential first step toward transforming the designed microscopic (molecular) property of the peptide-chromophore complex into a macroscopic material property. In addition, de noVo-designed peptides are typically more robust and stable than their natural counter-

Figure 1. (a) Chemical structure of 5,5′-bis[(10,20-di-((4-carboxymethyleneoxy)phenyl)porphinato)zinc(II)]butadiyne (referred to as PZnEEPZn). (b) Ruthenium(II) [5-(4′-ethynyl-(2,2′;6′,2′′-terpyridinyl))-10,20-bis(2′,6′-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2′;6′,2′′-terpyridine)2+ (referred to as Ru-PZn2+). (c) Peptide amino-acid sequences for H6H20 (AP0) and its variants, F6H20, H6F20, and F6F20.

parts while maintaining the designed functionality, making them ideal building blocks toward functional biomolecular materials.34,35 Here, we report our study on incorporating such extended chromophores into de noVo-designed amphiphilic peptides. Two zinc porphyrin-based extended chromophores were studied. 5,5′-bis[(10,20-di-((4-carboxymethyleneoxy)phenyl)porphinato)zinc(II)]butadiyne (PZnE-EPZn, previously referred to as “Zn33Zn”33) was chosen as a simple, symmetric linear, alkyne-bridged metal-porphyrin oligomer that possesses diminished potentiometric and optical band-gaps relative to simple monomeric (porphinato)metal compounds, and ruthenium(II) [5-(4′-ethynyl-(2,2′;6′,2′′-terpyridinyl))10,20-bis(2′,6′-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2′;6′,2′′-terpyridine)2+ (Ru-PZn2+) was chosen as an archetypal supermolecular nonlinear optical chromophore providing optimal alignment and coupling between (porphinato)metal and (terpyridyl)metal chargetransfer transition dipoles.9,19 The chemical structure of the PZnE-EPZn cofactor is shown in Figure 1a, and the synthesis and characterization were reported previously in the supporting information of ref 33. The chemical structure of the Ru-PZn2+ cofactor is shown in Figure 1b; the synthesis and characterization were reported in refs 9 and 19. The artificial protein scaffold was designed previously, based on a 4-helix bundle motif, is amphiphilic in overall nature and designated as AP0 (i.e., for Amphiphilic Protein 0).28 The peptide possesses both a hydrophilic and a hydrophobic domain along the length of its exterior, enabling the peptide’s 2388

insertion at an interface between a polar and nonpolar media. The interior of the protein scaffold was designed so that the prosthetic groups can be bound with axial ligation coordination chemistry at selected locations within the hydrophilic domain. AP0 and its variants are capable of binding metalloporphyrins via bis-histidyl axial coordination, although the (porphinato)zinc-based extended chromophores, PZnE-EPZn and Ru-PZn2+, are most likely only 5-coordinate (instead of 6-coordinate), utilizing only one of the two apposed histidyl residues for axial ligation. For both cases, incorporation of the extended conjugated chromophores into the 4-helix bundles did not interfere with the protein’s secondary structure. The cofactor binding occurs over broad ranges of ionic strength and surfactant concentration. The chromophore/peptide stochiometry can be tuned by varying the peptide oligomeric state and surfactant concentration. In the case of PZnE-EPZn, the binding affinities are comparable at different binding sites along the length of the bundle, enabling precise control of the exact location of the cofactor within the peptide scaffold. However, Ru-PZn2+ can bind only at certain locations along the bundle because of the steric hindrance of the (bisterpyridyl)ruthenium unit. The peptide/chromophore complexes are stable for months at room temperature and thermally stable up to 85 °C, maintaining more than 85% of the original helicity up to 80 °C. In an accompanying paper, we describe the assembly and structures of the peptide/chromophore complexes in Langmuir monolayers at the air/water interface.36 Briefly, both in the apo and holo forms (i.e., with Nano Lett., Vol. 6, No. 11, 2006

and without the extended chromophores, respectively) of the amphiphilic 4-helix bundles can be oriented vectorially in Langmuir monolayers at the air/water interface upon compression, as demonstrated by X-ray scattering and polarized absorption/emission spectroscopy. The 4-helix bundle structure was preserved upon incorporation of the extended chromophores. However, the monolayers of the vectorially oriented peptide/chromophore complexes exhibited only glass-like interbundle positional ordering in the monolayer plane. Shown in Figure 1c are the sequences of the amphiphilic peptides used. The originally designed AP0 possesses histidyl residues for bis-histidyl axial ligation of 6-coordinate metalloporphyrins between apposed helices in the 4-helix bundle at two binding sites separated by 14 residues along the length of the hydrophilic domain of the bundle (positions 6 and 20). We synthesized two variants of AP0 in which a His to Phe mutation was made at either position 6 or 20, to result in only one axial histidyl ligation site per helix, and designated these as F6H20 (i.e., H6-F6) and H6F20 (i.e., H20-F20). The mutations did not change the overall amphiphilic nature of the 4-helix bundle peptides, that is, the separate hydrophilic and hydrophobic domains along the length of the exterior of the bundles. Both peptides are capable of binding natural (porphinato) iron cofactors; such assemblies manifest redox potentials comparable to those evinced for natural heme proteins. A control peptide (F6F20) was also synthesized where both histidine (H6 and H20) positions were mutated to phenylalanine (F), thereby providing no metalloporphyrin binding sites along the helix. The chemical structure of the extended chromophore PZnE-EPZn is shown in Figure 1a. Note that 1,3-butadiyne-bridged PZnEEPZn can be rendered asymmetric in the 4-helix bundle peptide via axial histidyl ligation of only one of its two constituent Zn-porphyrin units. The chemical structure of the other extended chromophore, Ru-PZn2+, is inherently asymmetric, possessing only one (porphinato)zinc site for axial histidyl coordination (Figure 1b). The binding affinity of the maquettes for PZnE-EPZn was demonstrated via cofactor titration with the peptides as monitored by UV-vis absorption spectroscopy. Figure 2a shows a series of titration spectra upon adding the F6H20 di-helix (or dimer, see the Methods section) solublized in a buffered aqueous solution (50 mM potassium phosphate at pH ) 8) containing 0.9 wt % of the detergent n-octyl β-Dglucopyranoside (OG) into the PZnE-EPZn solution. Each spectrum was taken 1 min after adding an aliquot of the peptide to the PZnE-EPZn solution. Red spectral shifts from 422 to 424 nm, 478 to 483 nm, and 684 to 703 nm were clearly observed upon adding the peptide to the PZnE-EPZn solution. The peak intensities at 483 and 703 nm absorption maxima increase linearly upon titrating with peptide until the cofactor/peptide ratio reaches 1:4, after which there was no significant increase in absorbance. Titration experiments were also performed by titrating the cofactor (PZnE-EPZn) into the peptide solution, similar to previous titration experiments with (porphinato) iron componds.32 Spectral shifts toward the blue were observed upon adding increasing Nano Lett., Vol. 6, No. 11, 2006

amounts of PZnE-EPZn into the peptide solution. However, the overlapping of the different absorption peaks complicated the analysis and we chose to do the titration experiments in a “reverse fashion” as compared to previous heme binding experiments, that is, using the peptides to titrate the cofactors. Analysis of the spectra shows that the dissociation constant is less than 200 nM with a cofactor/peptide stoichiometry of 1 per 4-helix bundle.28 The dissociation constant was calculated using the absorption at 482 nm. The same experiments were performed using imidazole in place of the peptide, a heterocyclic compound with a structure similar to the side chain of histidine. Red spectral shifts (the peak positions are within 2 nm) similar to those of F6H20 were observed when imidazole ligates the PZnE-EPZn cofactor (data not shown). However, under the same experimental conditions, no spectral changes were observed for titrations with the control peptide, F6F20, where the histidine ligation sites were mutated to phenylalanine, thereby providing no metalloporphyrin binding sites along the helix. These results demonstrate that F6H20 is capable of binding the extended chromophore (PZnE-EPZn) specifically via axial histidyl ligation of one of its two (porphinato)zinc units at the H20 position in the 4-helix bundle peptide. The binding is most likely 5-coordinate utilizing only one of the four histidine residues available at that position in the 4-helix bundle (typical of Zn-porphyrins in natural proteins),37 in contrast to the 6-coordinate bis-histidyl coordination common for the b-type cytochromes. Figure 2b shows the titration spectra upon adding the H6F20 (dimer) solublized in the 0.9% OG buffered aqueous solution into the PZnE-EPZn solution. Red spectral shifts similar to those of F6H20 were observed, indicating specific binding of the PZnE-EPZn via axial histidyl ligation. The PZnE-EPZn binding affinity of H6F20 is very similar to that of F6H20. This is not the case for the natural cofactors such as Fe-porphyrins, where the dissociation constant at position 6 (H6) is typically 1 to 2 orders of magnitude lower than that of position 20 (H20). Fe-porphyrin titration experiments gave a dissociation constant of ∼50 nM for the H6F20 dimer and >1 µM for the F6H20 dimer (data not shown). Similar binding affinities at different locations along the length of the bundle for the (porphinato)zinc-based PZnE-EPZn enable us to control precisely the location of the PZnE-EPZn molecule within the maquettes via axial histidyl metal ligation. Thus, we can change the local dielectric environment to control the extent to which electron density is polarized in the extended chromophore, as well as the chromophore’s position relative to the exterior media. Figure 3 shows the schematic representations of the 4-helix bundle AP0 binding PZnE-EPZn at positions 6 and 20, respectively. For all cases, we expect the cofactor to be buried in the nonpolar interior of the 4-helix bundle (see accompanying paper).36 For position H6, only one possible structure then exists, as shown in Figure 3a, with the unligated Zn-porphyrin extending toward the interface between the hydrophilic and hydrophobic domains. For position H20, the same possible structure exists as shown in Figure 3a because the distance between the Zn atoms in the two porphyrins of the cofactor is 2389

Figure 2. (a) Titration of F6H20 into a 4 µM solution of PZnE-EPZn in a buffer solution (pH ) 8) containing 50 mM potassium phosphate and 0.9% (wt) OG. The spectrum was recorded in a 1 cm path length cuvette. The spectra shown contain 0, 1, 2, 3, 4, 5, 7, and 9 equiv of added peptide per cofactor. (b) Titration of H6F20 into 4 µM solution of PZnE-EPZn (50 mM KPi, 0.9 wt % OG, pH ) 8) recorded in a 1 cm path length cuvette. The spectra shown contain 0, 1, 2, 3, 4, 5, 6, and 7 equiv of added peptide per cofactor. Red spectral shifts from 422 to 424 nm, 478 to 483 nm, and 684 to 703 nm were clearly observed upon adding the peptide to the PZnE-EPZn solution.

comparable to the separation of the 6 and 20 positions along the length of the bundle. However, as shown in Figure 3b, the unligated Zn-porphyrin of the PZnEEPZn cofactor could also extend into the hydrophobic domain of the bundle, thereby spanning the interface between the hydrophilic and hydrophobic domains. We plan to employ resonance X-ray reflectivity, utilizing zinc as the resonant atom, to distinguish between these two possible structures for the F6H20 version of AP0. The binding of both (porphinato)zinc-based extended chromophores occurs over broad ranges of the ionic strengths 2390

and surfactant concentrations. The salt concentration can range from 50 to 400 mM, whereas the surfactant (OG) concentration of the peptide solution can span 0.9-4.5 wt %, with little effect on cofactor binding affinity. By varying the surfactant (OG) concentration and peptide oligomeric state, we can tune the chromophore/peptide stochiometry from 1 to 4 PZnE-EPZn per 4-helix bundle. Titration data show that the peptides solubilized in 0.9% OG buffer solution in the dimeric state (i.e., as covalently linked di-helices) can bind 1 PZnE-EPZn per 4-helix as shown in Figure 3, whereas the peptides solublized in 4.5% OG buffer solution remain Nano Lett., Vol. 6, No. 11, 2006

Figure 3. (a) Schematic representation of the cofactor PZnE-EPZn incorporated via axial histidyl ligation into the 4-helix bundle of H6F20 where the unligated Zn-porphyrin of the PZnEEPZn cofactor could also extend into the hydrophilic domain of the bundle. There are two possible orientations of the cofactor PZnE-EPZn incorporated into the 4-helix bundle of F6H20. The unligated Zn-porphyrin of the PZnEEPZn cofactor could extend into the hydrophobic domain of the bundle as shown in part b. The other possibility is that the unligated Zn-porphyrin of the PZnEEPZn cofactor extends into the hydrophilic domain of the bundle similar to what is shown in part a (see the text).

in the monomeric state and are capable of binding 4 PZnEEPZn per 4-helices. The peptides solubilized in 4.5% OG buffer solution in the dimeric state were capable of binding ca. 2-2.5 PZnE-EPZn per 4-helix bundle. This ability to tune the cofactor/peptide stochiometry may become important to increase the extended chromophore’s concentration

within oriented ensembles of the peptide/cofactor complexes while simultaneously controlling chromophore-chromophore interactions. Although there is no significant difference between the binding affinities at position 6 and position 20 along the length of the bundle for PZnE-EPZn, this is not the case for the Ru-PZn2+ cofactor. Both the designed amphiphilic peptide AP0 and its variant, H6F20, are capable of binding Ru-PZn2+. Figure 4 shows the titration spectra upon adding the H6F20 (dimer) solublized in the 0.9% OG buffered aqueous solution into the Ru-PZn2+ solution. Each spectrum was taken 1 min after adding an aloiquot of the peptide to the Ru-PZn2+ solution. Red spectral shifts from 428 to 431 nm, 509 to 515 nm, and 643 to 660 nm were clearly observed upon adding the peptide to the Ru-PZn2+ solution. The peak intensity at 660 nm increased linearly upon titrating the peptides until the cofactor/peptide mole ratio reaches 1:4, after which there was no significant increase in absorbance. The dissociation constant (calculated using the absorption at 660 nm) is less than 100 nM, comparable to that observed for PZnE-EPZn binding to this peptide. However, upon adding F6H20 (dimer) under the same conditions, we did not observe changes in the absorption spectrum of the cofactor. Binding did occur when adding F6H20 in the monomer form, but with the dissociation constant an order magnitude higher than that of H6F20 (data not shown). The de noVo-designed peptides, AP0 and its variants, are based on the 4-helix bundle motif. It is reasonable to speculate that upon incorporation of the extended PZnEEPZn chromophore, the 4-helix bundle formation will be distorted to some extent.36 The dependence of cofactor/ peptide stoichiometry on the surfactant concentration may well reflect the competition between 4-helix bundle formation and the incorporation of the extended chromophores. For the

Figure 4. Titration of H6F20 into a 3 µM Ru-PZn2+ in a buffer solution (pH ) 8) containing 50 mM potassium phosphate and 0.9 wt % OG. The series of spectra shown contain 0, 1, 2, 3, 4, 5, 6, and 7 equiv of added peptides per cofactor. Red spectral shifts from 428 to 431 nm, 509 to 515 nm, and 643 to 660 nm were clearly observed upon adding the peptide to the Ru-PZn2+ solution. Nano Lett., Vol. 6, No. 11, 2006

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Figure 5. Circular dichroism spectra of F6H20 in 50 mM potassium phosphate buffer solution (pH ) 8.0) containing 0.9% OG with (solid circles) and without (empty circles) PZnE-EPZn bound.

H6H20 AP0 peptide, the apo form is in an equilibrium mixture of dimer (i.e., 2-helix bundles) and tetramer (i.e., 4-helix bundles) at 0.9% OG, irrespective of whether or not the helices are dimerized to form covalently linked dihelices.28 Upon binding one Fe-porphyrin/4-helices via bishistidyl ligation, the equilibrium is shifted to 4-helix bundle formation, again irrespective of whether or not the helices are dimerized.28 We speculate that at a higher concentration of the surfactant OG, the AP0 peptides (H6H20 and its variants F6H20 & H6F20) do not form 4-helix bundles and, consequently, all of the hisitdyl binding sites are available for axial ligation. Because the titration experiments were necessarily performed by adding the peptides into the cofactor solution, the cofactors were generally in excess. The cofactors would tend to bind to all of the available sites. Thus, with the peptide in a monomeric state (i.e., as single helices) at 4.5% OG, the stochiometry is maximal at 4 per 4-helix bundle for the F6H20 & H6F20 peptides. In the case of the peptide dimers, the stochiometry reduces to 2-2.5 per 4-helix bundle, probably because of some steric hindrance. When the surfactant concentration is reduced to 0.9%, the formation of the 4-helix bundle is favored and, consequently, only one PZnE-EPZn can be incorporated into the 4-helix bundle. The fact that Ru-PZn2+ cannot bind to the F6H20 in a dimer form at 0.9% OG favoring 4-helix bundle formation supports the above speculation and is probably due to the bulkiness of the extended chromophore’s polypyridyl-ruthenium complex. We were unable to confirm the above, namely, that low detergent concentrations favor 4-helix bundle formation for the AP0 peptide variants (F6H20 and H6F20), using analytical ultracentrifugation because the peptides aggregated at such low detergent concentrations over the long centrifugation times required. However, grazing-incidence X-ray diffraction from Langmuir monolayers of the holo form of AP0 with the PZnE-EPZn cofactor at a cofactor-peptide stoichiometry of 1:4 with only minimal detergent and compressed to high surface pressures confirmed the presence of vectorially oriented 4-helix bundles at the air-water interface (see accompanying paper).36 The secondary structures of the AP0 and its variants were investigated using circular dichroism and were quite similar. Shown in Figure 5 is the CD spectrum of the F6H20 (dimer) that has minima at 222 and 208 nm, whose amplitudes 2392

Figure 6. Thermal denaturation of F6H20 with (solid triangles) and without (open circles) the cofactor PZnE-EPZn bound. The chromophore/peptide ratio is 1 PZnE-EPZn per 4-helix bundle. The peptide helicity was monitored at 222 nm. The sample was heated and equilibrated for 4 min at each temperature before the data was taken.

indicate >70% helical content. The intercalation of the extended chromophores PZnEEPZn into AP0 4-helix bundles (for variants H6H20, H6F20, and F6H20) and Ru-PZn2+ into AP0 (for variants H6H20 and H6F20) does not change the CD spectrum, indicating no significant perturbation of the secondary structure of the maquettes upon cofactor incorporation. All of the chromophore/peptide complexes are stable in buffered detergent solution at room temperature for months, although aggregation does occur at substantially lower temperatures (e.g., ∼4 °C), especially for minimal detergent concentrations. We did not observe precipitation of the complexes at room temperature as is frequently observed upon binding Fe-porphyrins to these same peptides. The thermal stabilities of the secondary structure of the maquette peptides in both the apo and holo form were studied by following the amplitude of the minimum at 222 nm in their CD spectra at elevated temperatures. Figure 6 shows the percentage of unfolded F6H20 (dimer) in the apo and holo forms upon heating. The maquettes maintained more than 85% of the original helicity up to 80 °C and are thermally stable both in the apo and holo form. The thermal stability of the PZnE-EPZn/peptide complexes was also monitored using UV-vis absorption spectra of the extended chromophores. Figure 7 shows two groups of UV-vis spectra. The first group (colored in blue) was obtained by heating the cofactor dissolved in the buffer solution (without peptides). No obvious spectral changes were seen except that the Q-band blue-shifted upon heating to 85 °C. These data suggest that at elevated temperature the distribution of PZnPZn interplanar torsional angles is broadened, resulting in a greater conformational heterogeneity, and augmented by an increased conformeric population weighted average PZnPZn torsional angle.10,22 Upon adding the peptide into the same cofactor solution (previously heated and subsequently cooled), the spectrum (colored in red) red-shifted, confirming that PZnE-EPZn was incorporated into the interior of the maquettes via axial histidyl ligation. The spectra did not change further upon heating to 85 °C, indicating that the PZnE-EPZn/F6H20 complex was stable up to that temperature. Compared with the pure cofactor solution (first group of spectra), we did not observe a blue shift of the Q-band Nano Lett., Vol. 6, No. 11, 2006

Figure 7. (a) UV-vis spectra of the F6H20/PZnE-EPZn complex at elevated temperatures, 25, 35, 45, 55, 65, 75, and 85 °C. The chromophore/peptide ratio is 1 PZnE-EPZn per 4-helix bundle. Each spectrum was taken after 10 min equilibration. (b) Expanded view of the UV-vis absorption spectra from 600-750 nm. For both parts, the series of spectra for the pure cofactor PZnE-EPZn in the buffer solution containing 0.9% OG upon heating is labeled with the reddish color. The series of spectra for the cofactor PZnE-EPZn upon specific binding to the F6H20 peptide is labeled with the blueish color. For both cases, the spectrum collected at higher temperature is labeled with a lighter shade as indicated by the arrow.

upon heating to 85 °C, indicating that incorporation of the cofactor into the 4-helix bundles constrains the PZnE-EPZn cofactor conformational heterogeneity to a narrower distribution of PZn-PZn interplanar torsional angles. At higher temperatures, the peptide/cofactor complexes started to denature and aggregate while the cofactors were no longer incorporated into the maquettes, as indicated by a blue shift in the Soret band of the spectra due to the presence of the unbound PZnE-EPZn. In conclusion, we demonstrate an effective method of controlling the local environment of porphyrin based extended chromophores that can be readily expanded to other similar systems, potentially leading to significant advances toward biomolecular-based nonlinear optical and electronic devices. In particular, we have reported the incorporation of two extended, conjugated, multipigment abiological chromophores into the de noVo-designed amphiphilic 4-helix bundle peptide AP0 and its variants. The (porphinato)zinc component of both PZnE-EPZn and Ru-PZn2+ chromophores was utilized to bind these cofactors via axial histidyl ligation at specific locations along the length of the interior of the 4-helix bundle with high binding affinities. Cofactor binding occurs over broad ranges of the ionic strengths and surfactant concentrations. The chromophore/peptide stochiometry can Nano Lett., Vol. 6, No. 11, 2006

be tuned from 1 to 4 cofactors per 4-helix bundle simply by varying the peptide oligomeric state via surfactant concentration. For the case of PZnE-EPZn, the binding affinities are comparable at different binding sites along the length of the interior of the 4-helix bundle. The chromophore/peptide complexes are thermally stable up to 85 °C and retain more than 85% of the original helicity up to 80 °C. Collectively, these attributes indicate that such de noVo-designed amphiphilic 4-helix bundle peptides are ideal candidates for the fabrication of nonlinear optical biomolecular materials. Methods. Synthesis. The peptides were synthesized on an Applied Biosystems Pioneer continuous flow solid-phase synthesizer using standard Fmoc/tBu protection strategy on a Fmoc-PEG-PAL-PS resin at 0.1 mmol scale. The peptides were acetylated at their N-terminus [1:1 (v/v) acetic anhydridepyridine for 3 min] and were cleaved from the resin and simultaneously deprotected using 90:8:2 trifluroacetic acid (TFA)/ethanedithiol/water for 3.5 h. Crude peptides were precipitated with cold ether and purified by reversed-phase C4 HPLC column chromatography using gradients of 6:3:1 2-propanol/acetonitrile/H2O and water containing 0.1% (v/ v) TFA. Pure peptides were dimerized by oxidizing the C-terminal cysteines in 1:1 (v/v) 100 mM ammoniumhydrogen carbonate buffer (pH 10.0) and methanol in air to form the 90 amino acid disulfide-linked di-helices and referred to as the peptide dimer. The peptide’s identity and purity were confirmed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF). The peptides were first dissolved in buffered aqueous solutions containing 4.5% (w/v) n-octyl beta-D-glucopyranoside (OG), 50 mM potassium phosphate at pH 8.0 and then diluted to final OG concentration of 0.9% (w/v). Peptide concentrations were determined by absorption at 280 nm because of the peptide’s single tryptophan residue assuming an extinction coefficient of 5690 M-1 cm-1, using PerkinElmer Lambda 2 spectrophotometer and a standard 1 cm path length quartz cuvette. The extinction coefficient was calculated from the primary sequence with the ProtParam tool offered by the EXPASY server of the Swiss Institute of Bioinformatics (http://us.expasy.org/cgi-bin/protparam).28 Detailed information on the synthesis of PZnE-EPZn was described previously.33 For the titration experiments, the cofactor was first dissolved in dimethyl sulfoxide (DMSO) as stock solution (2-6 mM) and diluted in a 0.9% OG, 50 mM potassium phosphate (pH ) 8) buffer solution. (The DMSO concentration is always lower than 0.1% in volume.) Peptide solution was added in 1 equiv aliquots to the cofactor and mixed well before the UV-vis spectra were taken. The values of cofactor dissociation constants were determined by monitoring UV-vis absorbance changes. For hightemperature UV-vis spectra, a heating cell with an accuracy of 0.1 °C was used. The solution was heated to a desired temperature and equilibrated for 10 min before recording each spectrum. Circular dichroism (CD) spectra were recorded with an Aviv 62DS spectropolarimeter using rectangular quartz cells of 2 mm path length for peptide concentrations of 30 µM. 2393

The sample was heated to an elevated temperature and equilibrated for 4 min. The helicity was measured at 222 nm. Acknowledgment. This work was supported by the Department of Energy grant DE-FG02-04ER46156 (M.J.T. and J.K.B.), and National Institutes of Health grants RR1481205 (T.X.) and GM-071628 (M.J.T.). J.K.B. and M.J.T. acknowledge the NSEC Program of the National Science Foundation (DMR-0425780) for infrastructural support. T.X. thanks S. Ye, B. M. Discher, K. Susumu, C. Moser, R. Koder, and P. L. Dutton for useful suggestions and discussions. References (1) Dalton, L. R.; Steier, W. H.; Robinson, B. H.; Zhang, C.; Ren, A.; Garner, S.; Chen, A. T.; Londergan, T.; Irwin, L.; Carlson, B.; Fifield, L.; Phelan, G.; Kincaid, C.; Amend, J.; Jen, A. J. Mater. Chem. 1999, 9, 1905. (2) Marder, S. R.; Kippelen, B.; Jen, A. K. Y.; Peyghambarian, N. Nature 1997, 388, 845. (3) Verbiest, T.; Houbrechts, S.; Kauranen, M.; Clays, K.; Persoons, A. J. Mater. Chem. 1997, 7, 2175. (4) Wolff, J. J.; Wortmann, R. AdV. Phys. Org. Chem. 1999, 32, 121. (5) Anderson, H. L. Chem. Commun. 1999, (23), 2323. (6) Anderson, H. L. Inorg. Chem. 1994, 33, 972. (7) Arnold, D. P.; Heath, G. A.; James, D. A. J. Porphyrins Phthalocyanines 1999, 3, 5. (8) Beljonne, D.; OKeefe, G. E.; Hamer, P. J.; Friend, R. H.; Anderson, H. L.; Bredas, J. L. J. Chem. Phys. 1997, 106, 9439. (9) Duncan, T. V.; Rubtsov, I. V.; Uyeda, H. T.; Therien, M. J. J. Am. Chem. Soc. 2004, 126, 9474. (10) Duncan, T. V.; Susumu, K.; Sinks, L. E.; Therien, M. J. J. Am. Chem. Soc. 2006, 128, 9000. (11) Lin, V. S. Y.; Dimagno, S. G.; Therien, M. J. Science 1994, 264, 1105. (12) Rubtsov, I. V.; Susumu, K.; Rubtsov, G. I.; Therien, M. J. J. Am. Chem. Soc. 2003, 125, 2687. (13) Screen, T. E. O.; Thorne, J. R. G.; Denning, R. G.; Bucknell, D. G.; Anderson, H. L. J. Am. Chem. Soc. 2002, 124, 9712. (14) Shediac, R.; Gray, M. H. B.; Uyeda, H. T.; Johnson, R. C.; Hupp, J. T.; Angiolillo, P. J.; Therien, M. J. J. Am. Chem. Soc. 2000, 122, 7017.

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