Computational design and elaboration of a de novo heterotetrameric a-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore

Article abstract of DOI:10.1021/ja907407m

The first example of a computationally de novo designed protein that binds an emissive abiological chromohore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C ₂-symmetric bundle, A_His:B_Thr, uniquely binds (5, 15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A₂B ₂ heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn: A _His:B_Thr]₂ is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A_His:B_Thr:(DPP)Zn protein (K _fluorescence = 4 x 10⁸ s^-1; τ_triplet = 5 ms). The A₂B₂ apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A_His:B_Thr protein ensure that interactions within the tetra-α-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors.

Full text of DOI:10.1021/ja907407m

Published on Web 03/01/2010
Computational Design and Elaboration of a de Novo
Heterotetrameric r-Helical Protein That Selectively Binds an
Emissive Abiological (Porphinato)zinc Chromophore
H. Christopher Fry,† Andreas Lehmann,†,¶ Jeffery G. Saven,*,†
William F. DeGrado,*,†,‡ and Michael J. Therien*,§
Department of Chemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6323,
Department of Biochemistry and Molecular Biophysics, Johnson Foundation, School of
Medicine, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6059, and Department
of Chemistry, Duke UniVersity, Durham, North Carolina 27708-0354
Received September 11, 2009; E-mail: saven@sas.upenn.edu; wdegrado@mail.med.upenn.edu;
michael.therien@duke.edu
Abstract: The first example of a computationally de novo designed protein that binds an emissive abiological
chromohore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This
heterotetrameric, C2-symmetric bundle, AHis:BThr, uniquely binds (5,15-di[(4-carboxymethyleneoxy)phe-
nyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The
A2B2 heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including
hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:
AHis:BThr]2 is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography,
and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data
reveal classic excited-state singlet and triplet PZn photophysics for the AHis:BThr:(DPP)Zn protein (kfluorescence
) 4 × 108 s-1; τtriplet ) 5 ms). The A2B2 apoprotein has immeasurably low binding affinities for related
[porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative
[(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally
designed protein. Importantly, elements of design in the AHis:BThr protein ensure that interactions within
the tetra-R-helical bundle are such that only the heterotetramer is stable in solution; corresponding
homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural
rearrangements that could lead to binding of (porphinato)iron cofactors.
Introduction
to craft tetra-R-helical peptides that bind abiological (porphi-
nato)iron (PFe) cofactors,10-12 elaborating entirely new met-
The design and synthesis of de noVo proteins that bind
abiological cofactors provide a general strategy to interrogate
important protein structure-function relationships1-5 and build
proteins that possess functions not seen in nature. Although de
noVo designed proteins capable of binding hemin [(PPIX)Fe]
have been realized,6-9 such systems typically mimic the
structures, functions and spectral features of naturally occurring
hemoproteins. While advances in protein design have been used
alloprotein functions requires the ability to engineer proteins
that uniquely accommodate targeted ligand frameworks as well
as specific metal ions. Designed protein complexes containing
abiological Zn-porphyrin cofactors are of particular interest due
to the fact that electron and energy transfer reactions can be
activated by pulsed laser excitation of such chromophores,13-17
(9) Ghirlanda, G.; Osyczka, A.; Liu, W. X.; Antolovich, M.; Smith, K. M.;
Dutton, P. L.; Wand, A. J.; DeGrado, W. F. J. Am. Chem. Soc. 2004,
126, 8141–8147.
† Department of Chemistry, University of Pennsylvania.
‡ Department of Biochemistry and Molecular Biophysics, Johnson
Foundation, School of Medicine, University of Pennsylvania.
§ Department of Chemistry, Duke University.
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M. J.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127, 1346–1347.
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D.; Therien, M. J.; Blasie, J. K.; Roder, H.; Saven, J. G.; DeGrado,
W. F. J. Am. Chem. Soc. 2007, 129, 10732–10740.
¶ Present address: Fox Chase Cancer Center, Philadelphia, PA 19111-
2434.
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A R T I C L E S
Fry et al.
and the fact that these moieties are key components in systems
exhibiting a variety of novel nonlinear optical and photophysical
properties.18-23 Herein, a helical bundle protein that selectively
encapsulates an emissive zinc porphyrin chromophore is com-
putationally designed and experimentally characterized.
and related proteins.37-42 Complete control and creation of a
protein environment that is complementary to an abiological
cofactor, however, might best be accomplished through auto-
mated de noVo design. Such approaches explicitly consider the
atomic details of the backbone structure as well side chain-side
chain and side chain-cofactor packing.
(Porphinato)zinc (PZn) chromophores have been utilized to
provide fundamental insights into electron and energy transfer
reactions13,16 and develop high performance electronic and
optical materials.18-23 Synthetic multiporphyrin arrays13,24-27
yield supramolecular architectures inspired in part by the
naturally occurring multiporphyrin array found in the light-
harvesting complex of photosystem II.28-30 While PZn may be
dispersed in polymer-based systems, controlling the cofactor’s
orientation and local environment can be difficult or limited.20,31,32
Though subtle, the folding and self-assembly properties of
proteins can be leveraged to arrive at well-defined local
environments for porphyrin-based cofactors. Bundles of helical
peptides can encapsulate and coordinate natural and abiotic Zn
porphyrins.33,34 Inspection of fiducial structures have guided the
design of such systems, where Zn is coordinated with appropri-
ate side-chain ligands (e.g., histidine) and hydrophobic residues
are patterned so as to stabilize a helical bundle. In addition,
such designed R-helical proteins can be displayed in oriented
molecular arrays at interfaces.34-36 Many of these systems
examined thus far have fluctuating tertiary structures, however,
and likely do not have the geometric complementarity and highly
structured physicochemical protein-porphyrin interactions ob-
served in natural proteins.33,35 Alternatively, non-natural (por-
phinato)metal derivatives have been incorporated into myoglobin
The de noVo design of soluble, proteins selective for PZn is
complicated by the preference of zinc porphyrins for a five-
coordinate environment.43-45 In contrast, the symmetry in six-
coordinate Fe3+ ligation can be leveraged in the design of
proteins that encapsulate iron porphyrins.10-12 Such preferred
local asymmetry in the metal coordination of PZn suggests use
of helical bundles having reduced symmetry, e.g., heterotet-
rameric bundles.46,47 Along these lines, heterotetramers have
been designed that present a dinuclear metal ion site48,49
including an A2B2 protein.50 Many previous bundle proteins
resulted from coarse-grained design approaches, including the
manipulation of complementary solvent-exposed electrostatic
interactions and empirical matching of interior hydrophobic side
chains.50 Heterotetrameric systems of this type have not been
identified for the much larger PZn cofactors.
Computational design methods provide powerful tools for
engineering structure and sequence.3,10-12,51-55 Herein, these
methods are applied to address specific protein-protein and
protein-PZn interactions in an atomically detailed manner.
Elements of both positive and negative design are used to arrive
at a de noVo heterotetrameric, R-helical peptide bundle that
selectively incorporates an abiotic PZn choromophore that can
be electronically excited to produce a long-lived fluorescent
state. We exploit the rigidity of tetra-R-helical peptides to
organize a (5,15-diarylporphinato)Zn cofactor [(DPP)Zn] at
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De Novo Metalloprotein Binds PZn Chromophores
A R T I C L E S
mM sodium chloride, pH 7.5; the protein concentration was 50
µM for the component peptides and 100 µM for the apo- and holo-
heterotetramers. (DPP)Zn was added from a 0.5 M NaOH stock
solution, as DMSO absorbs light in the 190-260 nm region of the
spectrum. All samples were filtered through a 0.22 µm spin filter.
For thermal denaturation experiments, the ellipticity at 222 nm was
monitored as a function of temperature. Data were collected every
2 °C; a heating rate of 1 °C/min was followed by a 4-min
equilibration time.
Stoichiometry. A solution of 2.5 µM (DPP)Zn was prepared
in 50 mM phosphate buffer, 150 mM sodium chloride, pH 7.5, 1%
octyl glucopyranoside (OG). (The surfactant, OG, aids in cofactor
solubilization.) Aliquots of AHis:BThr (0.50 µM) were introduced
into the solution and allowed to equilibrate for 5 min before a
spectrum was obtained. Spectrophotometric titrations were moni-
tored at 421 nm.
specified locations within an R-helical bundle. Previous designs
of helical peptides that bind abiological cofactors provided for
selective binding of (DPP)FeIII over iron(III) protoporphyrin
IX [(PPIX)FeIII].10-12 In contrast to the bis-His ligation
environment of (DPP)FeIII, the (DPP)Zn chromophore requires
mono-His coordination,33 which demands a less symmetric
protein environment relative to previous homotetrameric D2-
symmetric four-helix bundles. An appropriate A2B2 heterotet-
rameric protein is computationally designed and experimentally
characterized.
Experimental Methods
Synthesis of (DPP)Zn. 5,15-Di[((4-ethyl ester)methylene-oxy)-
phenyl]porphyrin. was synthesized according to a previously
published procedure.10
Job Analysis. Ten separate samples were prepared that varied
the ratio of the two peptides from 0:1f 1:1f1:0 AHis:BThr in 50
mM phosphate buffer, 150 mM sodium chloride, pH 7.5, (total
peptide] ) 150 µM). A 2-fold excess of the cofactor (DPP)Zn
was added from a concentrated DMSO stock solution, such that
the final DMSO concentration was kept below 1%. The sample
was heated to >50 °C for 15 min and then cooled to room
temperature. Any unbound cofactor formed visible aggregates that
were removed via spin filtration (0.2 µm). Each sample was
analyzed for cofactor binding via electronic absorption spectroscopy.
A Job plot monitoring peptide ratio versus cofactor absorbance at
550 nm was generated from the UV/visible spectral data.
Size Exclusion Chromatography. Gel filtration profiles were
obtained using a Superdex 75 10/300GL column on an FPLC
system (GE Healthcare AKTA FPLC System). To evaluate the
nature of the oligomeric state, 100 µL of a 250 µM sample was
injected onto the column and eluted with a flow rate of 0.5 mL/
min, and a 50 mM phosphate buffer, 100 mM NaCl, pH 7.5 mobile
phase. MWapp was calculated from a standard curve generated from
blue dextran (Vo), aprotinin (6500 Da), cytochrome c (12 400 Da),
carbonic anhydrase (29 000 Da), and albumin (66 000 Da) mass
standards. Results are provided in the Supporting Information.
Analytical Ultracentrifugation. For analytical ultracentrifuga-
tion, 125 µL samples of 600 µM AHis, BThr, AHis:BThr (1:1), and
25 µM (DPP)Zn:AHis:BThr in 50 mM phosphate buffer (100 mM
NaCl, pH 7.5) were prepared. The samples were filtered prior to
analysis using a 0.2 µm spin filter. The analysis was performed at
25 °C using a Beckman XL-I analytical ultracentrifuge. The
absorbance was monitored at 280 nm for peptide-only samples and
555 nm for samples containing the cofactor; the sample was
centrifuged at 35 000, 40 000, and 48 000 rpm. The data were analyzed
using a modified global fitting routine in IGOR Pro (Wavemetric).
The data were well-described by assuming a single molecular weight
species. The partial specific volume (ν) of 0.735 mL/g was calculated
using SEDNTERP (http://www.rasmb.bbri.org/).
[5,15-Di[((4-ethyl ester)methylene-oxy)phenyl]porphinato]zinc
[(DPPESTER)Zn]. In a round-bottom flask equipped with a condenser,
the free base porphyrin (126 mg, 0.19 mmol) was dissolved in 150
mL of chloroform. Zn(OAc)2 (340 mg, 2.1 mmol) was added to
the stirring solution and the mixture was refluxed for 3 h. The color
changed from a dark purple to an intense violet. The crude material
was obtained by removing the chloroform via rotary evaporation.
The product was purified by silica gel column chromatography (40:
60 THF/hexanes), which eluted as an intense violet band. The
solvent was removed under reduced pressure, yielding a purple
solid. Isolated yield ) 130 mg (95% based on 0.19 mmol of the
free-base starting material).
[5,15-Di[(4-carboxymethylene-oxy)phenyl]porphinato]zinc [(DP-
P)Zn]. (DPPESTER)Zn (130 mg) was saponified by refluxing with
KOH (90 mg, 1.6 mmol) in 80 mL of CHCl3/EtOH (3:1) for 3 h.
The solvent was removed under reduced pressure and redissolved
in water where the solution was acidified with 1 N HCl. The
precipitate was filtered, washed with 1 N HCl, dissolved in THF,
and then reprecipitated with hexanes. The supernatant was decanted
and the remainder of the solvent evaporated under reduced pressure,
yielding additional (DPP)Zn product. Isolated yield ) 118 mg (97%
1
yield based on 0.18 mmol of the ethyl ester starting material). H
NMR (CDCl3/1% pyridine-d5, 500 MHz): δ 10.5 (s, 2H), 9.6 (d,
4H), 9.3 (d, 4H), 8.3 (d, 4H), 7.7 (d, 4H), 5.4 (s, 4H). MS(MALDI-
TOF): m/z ) 672.52 (M+) (Calcd for C36H24N4O6: 672.10).
Peptide Purification. Two synthesized peptides AHis: Ac-
SLEEALQEIQ QAAQEAQQAL QKHQQALQKF QKYG-CONH2
and BThr: Ac-SLEEALQEAQ QTAQEFQQAL QKIQQAFQKF
QKYG-CONH2, were purchased from Biopeptide. The impure
material was further purified by reversed phase HPLC (Varian
ProStar) over a C4 column (Vydac). Analytical reversed phase
HPLC (Hewlett-Packard 1100) indicated successful purification of
the peptides. MALDI-TOF (PerSeptive Biosystems Voyager DE)
indicated correct mass spectral data (AHis Calcd, 3953.4; found,
3953.2. BThr Calcd, 4027.5; found, 4027.9).
Nanosecond Transient Absorption Spectroscopy. Transient
absorption spectra of 3 µM (DPP)Zn:AHis:BThr in 50 mM phosphate
buffer (100 mM NaCl, pH 7.5) were recorded with the previously
described Q-switched Nd:YAG laser (DCR-1A, Quanta Ray,
Mountain View, CA).56
General Sample Preparation. Samples were prepared by
introducing equimolar amounts of AHis and BThr to a 50 mM
phosphate buffer, 150 mM sodium chloride, pH 7.5. A 2-fold excess
of the cofactor (DPP)Zn was added from either a 5 mM DMSO or
0.5 M NaOH stock solution. (Note: final DMSO concentration was
kept below 1%.) The sample was heated to >50 °C for 15 min,
followed by cooling to room temperature. Any unbound cofactor
precipitated under these conditions, which was removed via spin
filtration (Millipore Ultrafree MC 0.22 µm).
UV-Visible and Circular Dichroism (CD) Spectroscopy. To
verify binding of the cofactor, complex formation was monitored
at the Soret band at 420 nm and Q-band at 551 nm. Electronic
absorption spectra were obtained on a Hewlett-Packard 8453
UV-visible spectrophotometer at room temperature in 1-cm quartz
cells. To evaluate the backbone conformation and stability of the
protein-cofactor complex, CD spectroscopy was carried out at 25
°C using a Jasco 810 spectropolarimeter in 0.1-cm cells. Buffer
conditions for CD experiments were 50 mM phosphate buffer, 150
Picosecond Fluorescence Lifetime Measurements (Streakscope).
Time-resolved emission spectra of 3 µM (DPP)Zn:AHis:BThr in 50
mM phosphate buffer (100 mM NaCl, pH 7.5) were recorded using
a Hamamatsu C4780 picosecond fluorescence lifetime measurement
system. This system employs a Hamamatsu Streakscope C4334 as
its photon-counting detector and a Hamamatsu C4792-01 synchro-
nous delay generator. A Hamamatsu 405 nm diode laser served as
the excitation light source. All fluorescence data were acquired in
single-photon-counting mode using Hamamatsu HPD-TA software.
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A R T I C L E S
Fry et al.
Figure 1. Model of the designed protein illustrating (A) the AHis:BThr:(DPP)Zn structure, (B) residues near the coordinating H22, (C) details of four residue
10-12
modifications on the distal side, and (D) the final sequences compared to that of PATet
.
The data were analyzed using a single exponential kinetic fit in
OriginLab (7.5).
indicated, the computationally determined amino acid identity
applies to both peptides. At the underlined positions the most
probable amino acid identities were selected. E7 and E14 form
complementary electrostatic interactions with positions 21 and 28,
positions that were subsequently constrained to allow only positively
charged amino acids (K, R). Subject to these constraints on
sequence, a second calculation was performed. The resulting
probabilities were used to specify E2, L5, A12, L19, K21,
A25_(AHis), K28, K31, and Y32. A12 was the second-most probable
identity in position 12, which was selected over S since it resides
at the interhelical interface. K was the third-most probable identity
in position 31, an exterior position at which many amino acids have
comparable probabilities. Before the third calculation, at all
remaining variable exterior positions, ionizable amino acids and
proline (D, E, H, K, P, and R) were precluded so as to not frustrate
predetermined interhelical electrostatic interactions. After this third
calculation, the remaining identities were fixed: Q6, Q9, Q10, A11
(AHis), Q13, Q16, Q17, Q20, I22 (BThr), Q23, Q24, A25 (BThr),
L26 (AHis), Q27, and Q30. All of these selections, except A25,
were the most probable choices at these respective positions. The
third-most probable A25 (BThr), was chosen over the most probable
G and the second-most probable S, to support an interhelical
hydrophobic zipper motif involving I8 (AHis).
Figure 1 provides a comparison of the newly designed sequences
(PZn peptide) with an earlier (DPP)Fe design (parent tetramer
PATet) of Cochran et al.10,11 Among the 64 unique residue positions,
the (DPP)Zn design differs from the previous (DPP)Fe protein at
five positions. Two mutations, T11A (AHis) and H22I (BThr), result
from changing the metal environment from six-point to five-point
coordination; these residues are distal to the coordinating His,
effectively removing the second coordination site while maintaining
a hydrophobic interior (Figure 1). Mutations A8I (AHis) and F26L
(AHis) are sterically linked to the mutation H22I (BThr). Mutation
A15F (BThr) results from the reduced symmetry compared to the
Computational Methods
Requirements for efficient cofactor binding dictated the protein
backbone geometry as described previously in the design of an iron-
porphyrin binding protein.10 Zn2+ prefers a pentacoordinate envi-
ronment, and only one axial His ligand was specified to coordinate
each cofactor, yielding a C2 symmetric structure. The resulting A2B2
tetramer contains two distinct peptides, labeled AHis and BThr; the
two AHis helices are antiparallel to one another as are the two BThr
helices. AHis contributes a Zn-coordinating His, and BThr contributes
a Thr that is poised to interhelically hydrogen bond with the His
of AHis. The identities of these two residues (His and Thr) are
constrained within the tetrahelical structure, and those of the
remaining 62 variable positions are determined using computational
sequence design. In Zn-porphyrins, as opposed to ferric-porphyrins,
the Zn ion is slightly domed out of the plane defined by the
porphyrin macrocycle (see Table S1 of Supporting Information).
The sequence calculations described below were found to be
insensitive to dome heights in the range 0.0-0.3 Å.
Protein Sequence Design. The identities of the 62 variable
positions were determined by recursive computational design
calculations of the probabilities of the amino acids at variable
positions.3,57 This statistical, computationally assisted design
strategy (SCADS) has been used previously.3,10,58-60 Seventeen
natural amino acids were allowed at the variable positions. Cysteine,
histidine, and proline were excluded, as was methionine at positions
facing the protein interior; proline has a low helix propensity, and
histidine, cysteine and methionine could introduce nontargeted
metal-coordination. Positions 11 in BThr and 22 in AHis were
constrained to be Thr and His, respectively. Three rounds of
calculations were performed. In the first round, hydrophobic residues
A, F, G, I, L, V, and W were allowed in the interior positions 5, 8,
15, 19, 26, and 29. The site-specific probabilities of the amino acids
were determined as described previously.10,11 After the first round,
identities L1, E3, A4, E7, I8 (AHis), A8 (BThr), E14, A15(AHis),
F15 (BThr), A18, F26 (BThr), and F29 were specified. Selections
were made from the probable amino acids at each site, and unless
previous homotetramer. A8I (AHis), F26L(AHis), and A15F(BThr
)
yield a protein interior that is both more well-packed and more
hydrophobic than the previously designed (DPP)Fe protein.10,11
Results
Protein Design. Requirements for efficient cofactor binding
dictated the protein backbone geometry as described previously
in the design of a homotetrameric precursor bundle (PAtet) that
bound ferrous and ferric DPP cofactor in a bis-His geometry.10
Zn2+ prefers a pentacoordinate environment, and only one axial
His ligand was specified to coordinate each cofactor, yielding
a C2 symmetric structure. Following a minimalist strategy, the
sequences of the two helices, designated AHis and BThr, were
(57) Kono, H.; Saven, J. G. J. Mol. Biol. 2001, 306, 607–628.
(58) Swift, J.; Wehbi, W. A.; Kelly, B. D.; Stowell, X. F.; Saven, J. G.;
Dmochowski, I. J. J. Am. Chem. Soc. 2006, 128, 6611–6619.
(59) North, B.; Cristian, L.; Stowell, X. F.; Lear, J. D.; Saven, J. G.;
DeGrado, W. F. J. Mol. Biol. 2006, 359, 930–939.
(60) Nanda, V.; Rosenblatt, M. M.; Osyczka, A.; Kono, H.; Getahun, Z.;
Dutton, P. L.; Saven, J. G.; DeGrado, W. F. J. Am. Chem. Soc. 2005,
127, 5804–5805.
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4000 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

De Novo Metalloprotein Binds PZn Chromophores
A R T I C L E S
Chart 1
Figure 2. UV/vis spectra of DPP(Zn), 2.5 µM in 50 mM phosphate buffer,
150 mM NaCl, pH 7.5 (solid black line), with 1% w/v octylglucopyranoside
(dashed black line), and in the presence of 1:1 AHis:BThr (solid red line)
with 1% w/v octylglucopyranoside.
designed to introduce the desired specificity; the peptides should
bind DPP(Zn) only when both the AHis and BThr peptides are
present as a binary mixture. In the original homotetrameric PAtet
bundle, each helix contains a single His and Thr residue. Thr
and His residues on adjoining helices form a network of
interhelical second-shell hydrogen bonds that create two sym-
metry-related bis-His binding sites appropriate for interaction
with ferrous and ferric porphyrin derivatives. In contrast, the
AHis and BThr helices of the current design have a single His
and Thr, respectively. The resulting (AHis:BThr)2 heterotetramer
retains the interhelical second-shell hydrogen bonds of PAtet,
but due to the lack of a His residue in BThr, two single-His
sites are formed to provide specificity for pentacoordinate Zn(II)
porphyrins. Moreover, the side-chain packing algorithm showed
that specific buried positions had a clear preference for large
apolar side chains that could not be accommodated in the starting
PAtet sequence, due to steric overlap when all four chains of
the protein were forced to have the same sequence. The relaxed
symmetry of the heterotetrameric bundle allowed positioning
of these larger apolar side chains in a geometrically comple-
mentary manner, potentially increasing the stability of the
complex, as well as the specificity for binding to the heterotet-
rameric bundle, versus potential homo-oligomers of the con-
stituent individual peptides. In summary, the AHis:BThr het-
erotetramer should bind specifically to two DPP(Zn) cofactors
only if the two peptides were present at a 1:1 stoichiometry,
and with the helices in an antiparallel arrangement (a parallel
bundle would position the two His residues on the same side,
leading to a bis-His site for a single cofactor, or two side-to-
side sites leading to negative cooperativity, with the second
binding porphyrin more weakly than the first).4,33
Figure 3. Titration of 1:1 AHis:BThr mixture into a 2.5 µM DPP(Zn) solution
in 50 mM Pi, 150 mM NaCl, pH 7.5 and 1% OG surfactant (w/w). Inset:
binding curve monitored at 420 nm fit with a dissociation curve. Dashed lines
are included in the inset plot of absorbance vs [AHis:BThr ]/2[DPP(Zn)] to
indicate a stoichiometry of 1:1:1 AHis:BThr:DPP(Zn).
intensities; upon addition of surfactant (1 wt % OG), B-state
(λmax(B-state) ) 411 nm) and Q-state absorption (λmax(Q-state)
) 545 nm) maxima are evident. When a 1:1 AHis:BThr peptide
mixture is added, both bands red-shift (λmax(B-state) ) 421 nm,
λ
max(Q-state) ) 551 nm), consistent with His ligation to the
DPP(Zn) cofactor. Electronic absorption spectroscopy was
utilized to monitor titrations of protein into DPP(Zn) solutions,
which demonstrates a 1:1:1 [DPP(Zn):AHis:BThr] cofactor:
protein complex stoichiometry, as expected for mono-His
coordination of DPP(Zn) (Figure 3). Addition of protein in
excess of the stoichiometric amount does not further augment
the B band absorbance. The titration data of Figure 3 may be
fit with a two-site binding model having values of the dissocia-
tion constants for the first and second equivalents of the cofactor
of K1 ) 10 µM and K2 ) 8 × 10-3 µM, respectively (see
Supporting Information). Such estimates are approximate and
nonrigorous thermodynamically due to the poor solubility of
the cofactor and the presence of detergent in the samples.
Notwithstanding, this analysis provides an empirical description
of the binding process that demonstrates (1) a high cooperativity
to the assembly process (the first cofactor is bound weakly, while
the second is bound tightly); (2) the mean overall dissociation
constant, (K1K2)1/2 ) 0.28 µM, is in the submicromolar range
(and consistent with values found for a five-coordinate heme
protein maquette).7,61 Ratiometric titrations (Job plot analysis)
involving the two peptides (AHis:BThr 1:0 f 1:1 f 0:1) evince
Cofactor. (DPP)Zn (Chart 1) shows two pyrrole peaks that
1
appear at 9.6 and 9.3 ppm in the H NMR data (d6-DMSO)
suggesting D2-symmetry. This cofactor in buffer (50 mM
phosphate, 150 mM NaCl, pH 7.5) is not readily soluble and
aggregates appreciably, resulting in broad UV/visible bands in
the Soret and Q-band regions of the spectrum (Vide infra). For
clarity, Chart 1 shows the zinc protoporphyrin IX ((PPIX)Zn)
structure; note that the nature and position of the (PPIX)Zn
peripheral macrocycle substituents (ꢀ-CH3, CHCH2, and
-CH2COO-) differ markedly from those of DPP(Zn) (meso-4′-
carboxymethyleneoxyphenyl).
Electronic Absorption Spectroscopy. In the absence of
peptide, the cofactor partially aggregates, resulting in broad,
red-shifted B- and Q-band manifolds (Figure 2) having low
(61) Petros, A. K.; Shaner, S. E.; Costello, A. L.; Tierney, D. L.; Gibney,
B. R. Inorg. Chem. 2004, 43, 4793–4795.
9
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A R T I C L E S
Fry et al.
Figure 4. Job plot (inset, λprobe ) 550 nm) and corresponding DPP(Zn)-
based electronic absorption spectral dependence upon the AHis:BThr molar
ratio. Experimental conditions: [DPP(Zn)] ) 75 µM, [total peptide] ) 150
µM, 50 mM Pi, 150 mM NaCl, pH ) 7.5.
Figure 6. UV/vis spectra of (DPP)FeIII (5 µM in 50 mM Pi, 150 mM
NaCl, pH 7.5) in the presence of AHis (red), 1:1 AHis:BThr (purple), and
B
Thr (blue) (total peptide concentration, 10 µM). Inset: Job plot monitored
at 408 nm, indicating no binding of the cofactor to the peptide mixture.
Figure 5. UV/vis spectra of 5 µM (PPIX)Zn in 50 mM Pi, 150 mM NaCl,
pH 7.5 (black), 5 µM (PPIX)Zn:AHis:BThr (red), and 5 µM (PPIX)Zn with
25 µM AHis and 25 µM BThr (blue).
a maximal DPP(Zn) binding efficiency at 1:1 AHis:BThr (Figure
4, inset), consistent with the targeted structure of the complex.
Homogeneous peptide solutions of either AHis or BThr do not
bind DPP(Zn) as indicated by the overall low absorption
intensity in the visible spectrum, consistent with aggregated,
unbound cofactor (Figure 4).
The designed heterotetrameric PZn-binding protein is por-
phyrin macrocycle selective. UV-visible spectroscopic studies
probing cofactor specificity show that 1:1 solutions of AHis
:
Figure 7. (A) Normalized circular dichroism spectra (25 °C) of AHis (red),
BThr (blue), AHis:BThr (1:1 mixture, black), and (DPP)Zn:AHis:BThr(1:1:1
mixture, purple); the inset shows the corresponding visible/Soret spectrum.
(B) Thermal unfolding profiles (λprobe ) 222 nm) of AHis (red, TM ) 44
°C), BThr (blue, TM ) 54 °C), AHis:BThr (black, TM ) 56 °C), and complexed
(DPP)Zn:AHis:BThr (purple, TM ) 63 °C). The fraction folded was estimated
using the ellipticity relative to folded and unfolded baselines.
B
Thr do not bind (PPIX)Zn as indicated by the absence of any
significant change in the absorption spectrum, particularly the
intensity of the B-band at 420 nm (see Figure 2), upon exposure
to the designed peptides (Figure 5). In addition, B-band (423
nm) and Q-band maxima (549-550 nm and 585-587 nm) were
not observed, as has been reported for bound (PPIX)Zn
analogues of hemoglobin62 and cytochrome c.63 Interestingly,
similar studies64 as well as Job analysis (Figure 6) indicate that
(DPP)FeIII is not bound at any AHis:BThr molar ratio, including
homogeneous peptide solutions where a 2:1 AHis:(DPP)Fe
stoichiometry is maintained (Figure S1, Supporting Information),
congruent with the homomeric bundles of AHis being under-
packed and unstable. This sophisticated level of cofactor
differentiation contrasts sharply with the properties of a ligation
site in a helix-turn-helix peptide dimer that bound either two
(PPIX)Zn species with mono-His coordination or one (PPIX)Fe
cofactor in a classic bis-His mode.33
CD Spectroscopy. CD spectroscopy confirms varying degrees
of R-helical secondary structure for the individual units of AHis
and BThr, as well as the 1:1 mixture AHis:BThr and AHis:BThr
:
(DPP)Zn (Figure 7A) as indicated by the minima at 208 and
222 nm.65 A binary 1:1 mixture of AHis:BThr showed a CD
spectrum typical of a helical protein. No change was observed
upon addition of stoichiometric (DPP)Zn cofactor, again
supporting the conclusion that the apoprotein was already folded
(62) Leonard, J. J.; Yonetani, T.; Callis, J. B. Biochemistry 1974, 13, 1460–
1464.
(63) Vanderkooi, J. M.; Adar, F.; Erecinska, M. Eur. J. Biochem. 1976,
64, 381–387.
(64) This result is in contrast to the parent homotetramer which binds both
Fe(DPP) and Zn(DPP) (results not shown).
(65) Pescitelli, G.; Gabriel, S.; Wang, Y. K.; Fleischhauer, J.; Woody,
R. W.; Berova, N. J. Am. Chem. Soc. 2003, 125, 7613–7628.
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4002 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

De Novo Metalloprotein Binds PZn Chromophores
A R T I C L E S
Figure 8. Sedimentation equilibrium of (DPP)Zn:AHis:BThr (30 µM, RT)
at various angular velocities (35, red; 40, green; and 48 kRPM, blue)
monitoring the absorbance at 555 nm (Q-band of (DPP)Zn) yields an
appropriate MW for the bundle, 17265 ( 200. MWcalc ) 17309.8 (ꢁ2 )
3.06). The residuals are shown in the top panel; they were random within
the experimental error of the measurement and showed no systematic errors.
in the absence of cofactor. The enhanced stability of apo-AHis
:
BThr is likely due in part to its superior hydrophobic interior
packing.
Although the (DPP)Zn cofactor is achiral, it becomes
optically active when bound in the chiral environment of the
AHis:BThr tetramer. The visible region of the spectrum indicates
a positive Cotton effect for the two PZn chromophores with
λmin ) 423 nm and λmax ) 416 nm. This observation suggests
that the two (DPP)Zn chromophores are aligned in a specific
rigid conformation along the superhelical axis of the bundle; in
the absence of peptide, no (DPP)Zn CD signal is observed.65,66
Figure 9. Time resolved fluorescence of AHis:BThr:(DPP)Zn in 50 mM
Pi, 150 mM NaCl, pH 7.5 buffer, λex ) 405 nm. (A) Fluorescence spectra
obtained at various time delays (black, <1 ns; red, 1 ns; blue, 2 ns; green,
3 ns; orange, 4 ns; yellow, 10 ns). (B) The kinetic profile obtained from
the time-dependent integrated emission over the 600-700 nm spectral
region; the red line is a first-order fit, yielding a time constant of 4 × 108
The stabilities of the component peptides, AHis:BThr and AHis
:
s-1
.
B
Thr:(DPP)Zn were further examined by monitoring their
thermal unfolding profiles (Figure 7B). Although the unfolding
curves were not fully reversible, precluding a thermodynamic
analysis, they provide qualitative data concerning the stabilities
of the various assemblies. The BThr peptide had greater stability
than the AHis peptide, and when mixed, the sample unfolded in
a single transition with a midpoint near that of the BThr sample.
Thus, addition of BThr leads to the formation of a complex in
which AHis is substantially stabilized toward denaturation.
Furthermore, the addition of stoichiometric (DPP)Zn further
increased the midpoint by approximately 7 °C, indicative of
specific binding to the heterotetrameric protein.
formed, MWcalc (DPP)Zn:AHis:BThr ) 17309.6; MWfound
(DPP)Zn:AHis:BThr ) 17265 ( 200. Size exclusion chroma-
tography confirmed the association states of the peptides in the
absence of the cofactor, but the chromatography matrix inter-
acted with (DPP)Zn, leading to problems with reproducibility
and difficulties in the interpretation of the results for the holo-
protein.
Photophysical Characterization. Steady-state emission, as
well as time-resolved transient absorption and fluorescence
spectroscopic data, demonstrate classic excited-state singlet and
triplet PZn photophysics for the AHis:BThr:(DPP)Zn protein.
Equilibrium Analytical Ultracentrifugation (AUC). AUC was
employed to determine the oligomerization states of the
individual peptides, AHis and BThr; the 1:1 mixture of the
AHis:BThr:(DPP)Zn displays S1 f S0 fluorescence emission
(λem(max) ) 641 nm) typical of its protein-bound chromophore
(Figure S2, Supporting Information). Time resolved experiments
(λex ) 405 nm) yield a fluorescence decay time constant (kf )
4 × 108 s-1) similar to that exhibited by Zn cytochrome c (S1
f S0, λem ) 595, 650 nm; kf ) 3 × 108 s-1), Figure 9.67
Pump-probe transient absorption spectroscopy (λex ) 532 nm)
peptides, AHis:BThr; and the fully formed bundle, (DPP)Zn:AHis
:
BThr (Figure 8). AUC for AHis suggests trimer formation: MWcalc
[AHis]3 ) 11860.2; MWfound [AHis]3 ) 11731 ( 153. Similarly,
AUC analysis for BThr is consistent with tetramer formation:
MWcalc [BThr]4, 16110; MWfound [BThr]4, 16462 ( 188. For a
1:1 mixture of AHis:BIle (apo form), the computed molecular
weight was slightly higher than that expected for the heterotet-
rameric bundle (MWcalc ([AHis]:[BThr])2 ) 15960; MWfound
([AHis]:[BThr])2 ) 18797 ( 322), suggestive of a small amount
of higher-order aggregation or lack of accuracy in the computed
partial specific volumes used to convert the buoyant MW to
the observed MW. In contrast, upon incorporation of the
cofactor, a homogeneous tetra-R-helical holoprotein sample was
indicates high quantum yield for relaxation of AHis:BThr
:
1(DPP)Zn* to its corresponding triplet state, AHis:BThr:3(DPP)-
Zn *, Figure 10. The measured triplet lifetime of the electroni-
cally excited holoprotein, τtriplet ) 5 ms, is consistent with similar
lifetime determinations of the photoexcited triplet states of PZn
compounds in organic solvents (τtriplet ) 1.2 ms),68 as well as
with that evaluated for Zn cytochrome c (τtriplet ) 14 ms).67
(67) Sudha, B. P.; Dixit, N.; Moy, V. T.; Vanderkooi, J. M. Biochemistry
1984, 23, 2103–2107.
(66) Guryanov, I.; Moretto, A.; Campestrini, S.; Broxterman, Q. B.; Kaptein,
B.; Peggion, C.; Formaggio, F.; Toniolo, C. Biopolymers 2006, 82,
482–490.
(68) Darwent, J. R.; Douglas, P.; Harriman, A.; Porter, G.; Richoux, M. C.
Coord. Chem. ReV. 1982, 44, 83–126.
9
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A R T I C L E S
Fry et al.
design A2B2 was engineered such that each histidine would be
associated with a single (DPP)Zn chromophore. The design
departed from the previously reported PATet, which bound two
equivalents of a (DPP)Fe cofactor using a bis-His coordination
site.10 The calculations identified isoleucine as an appropriate
amino acid to position opposite the coordinating histidine. Red
shifts in the B- and Q-bands of the UV/visible spectrum provide
evidence of (DPP)Zn binding to a histidine side chain, which
is consistent with the 1:1:1 AHis:BThr:(DPP)Zn stoichiometry
of association observed in the spectrophotometric titrations.
Finally, the ratiometric titration suggests a 1:1 mixture is
required to bind the (DPP)Zn cofactor. We are aware of only
one other designed peptide capable of binding a (porphinato)zinc
cofactor - a homodimeric tetra-R-helical bundle that is known
to undergo a structural rearrangement when it binds a (porphi-
nato)iron compound.33 The tetra-R-helical system reported
herein does not only preferentially bind (DPP)Zn with respect
to its corresponding (porphinato) iron derivative; the AHis:BThr
apoprotein is designed in a manner that bars structural rear-
rangement that would produce a stable bis-His coordination
environment for the equivalent Fe-containing cofactor analogue,
(DPP)Fe (Vide infra).
Controlled Organization of Bundle. We employed the statisti-
calcomputationallyassisteddesignstrategy(SCADS)algorithm57,76
to identify an appropriate heterotetrameric (A2B2) bundle similar
to the work with the homotetrameric (DPP)FeII/III-binding
protein10 (Figure 1). The final sequences reflect ligand-directed
elements of both positive and negative design and a mix of
interactions that include hydrogen-bonds to second-shell ligands.
One peptide chain, AHis contains the His ligand while chain BThr
contains its complementary secondary ligand, Thr. An implicit
element of negative design in this arrangement is that this
interaction can form only in the heterotetramer; homomeric
bundles thus present an unstable ligand-binding geometry. A
second element of positive design is the enhanced interior
packing associated with A16F mutation on BThr and F26L on
Figure 10. (A) Transient absorption difference spectra of AHis:BThr
:
(DPP)Zn in 50 mM Pi, 150 mM NaCl, pH 7.5 buffer, λex ) 532 nm. (B)
The kinetic profile determined at 480 nm of the corresponding Figure 9A
data (black dots); a fit to two exponential processes (red line) gives τ1 )
175 µs and τ2 ) 4.5 ms.
A
His; these side chain replacements also serve as a negative
The triplet lifetime of PZn compounds fully exposed to water
is τtriplet ) 0.1-0.4 ms, suggesting that the protein sequesters
the cofactor from solvent exposure.69
design element in that the homotetramer formed from the AHis
chains is underpacked in these regions, while the corresponding
homogeneous BThr bundle shows repulsive steric interactions
at Phe16. A third element of positive design is the identification
of interior side chains that form a largely hydrophobic cavity
tailored to the (DPP)Zn cofactor.
All three elements of positive and negative design were
checked by a Job analysis with the (DPP)FeIII synthetic cofactor.
The ferric center prefers a six coordinate geometry, therefore 4
Discussion
A tetrameric R-helical bundle capable of binding an abio-
logical (porphinato)zinc cofactor has been successfully designed
and characterized. This protein binds the cofactor via a
combination of designed shape complementarity, electrostatic
interactions, and a single histidine to coordinate the Zn. The
computationally designed protein assembly forms the predicted
bundle when all three components (AHis, BThr, and (DPP)Zn)
are present, and the targeted peptide stoichiometry has the
highest affinity for the cofactor. The protein selectively incor-
porates the targeted (DPP)Zn chromophore and binds neither
(DPP)Fe nor (PPIX)Zn (Zn-hemin). In this work, computational
protein design has been used to organize two synthetic emissive
PZn chromophores, in a targeted spatial arrangement and
molecular environment, that is selective for both metal ion and
the nature of the peripheral porphyrin macrocycle substituents.
Five-Coordinate Geometry. (Porphinato)zinc compounds are
known to bind a single axial ligand.70-75 The heterotetrameric
AHis peptides should provide a bis-His ligation environment in
a homotetrameric bundle. However, binding of (DPP)FeIII was
not observed, presumably due to the lack of the hydrogen-
bonding residue, Thr8. In addition, the lack of bundle formation
when mixing 4 AHis + (DPP)FeIII can be directly related to the
lack of interhelical and core Phe residues present in the previous
homotetramer;10 the absence results in significant underpacking
of the 4 AHis homotetramer. Furthermore, a 1:1 ratio of AHis
:
(72) Barkigia, K. M.; Berber, M. D.; Fajer, J.; Medforth, C. J.; Renner,
M. W.; Smith, K. M. J. Am. Chem. Soc. 1990, 112, 8851–8857.
(73) DiMagno, S. G.; Lin, V. S.-Y.; Therien, M. J. J. Org. Chem. 1993,
58, 5983–5993.
(74) Goll, J. G.; Moore, K. T.; Ghosh, A.; Therien, M. J. J. Am. Chem.
Soc. 1996, 118, 8344–8354.
(69) Kim, J. E.; Pribisko, M. A.; Gray, H. B.; Winkler, J. R. Inorg. Chem.
2004, 43, 7953–7960.
(75) LeCours, S. M.; DiMagno, S. G.; Therien, M. J. J. Am. Chem. Soc.
1996, 118, 11854–11864.
(76) Park, S.; Xi, Y.; Saven, J. G. Curr. Opin. Struct. Biol. 2004, 14, 487–
494.
(70) Scheidt, W. R.; Lee, Y. J. Struct. Bonding (Berlin) 1987, 64, 1–70.
(71) Collins, D. M.; Hoard, J. L. J. Am. Chem. Soc. 1970, 92, 3761–71.
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De Novo Metalloprotein Binds PZn Chromophores
A R T I C L E S
B
Thr provides an appropriate ligand environment for (DPP)Zn,
This work offers an alternative and important complement
to synthetic multiporphyrin arrays that utilize covalent and/or
coordinative interactions between metalloporphyrin units to drive
modest degrees of chromophore-chromophore electronic
coupling.13,24-26 Protein design offers an approach to achieve
specific spatial arrangements of cofactors in a completely water-
soluble system; furthermore, protein surface functionality can
in principle be exploited to direct higher levels of organization
of these electrooptically addressable components. As peptide
design strategies become increasingly more sophisticated,
covalent connections between cofactors that enable strong
coupling will yield systems that offer entirely new electrooptical
functionality in protein environments.18-23
but a single histidine for (DPP)FeIII renders the d5 transition
metal coordinatively unsaturated. These design features present
in a 1:1 AHis:BThr ratio preclude rearrangements to accommodate
a ferric porphyrin cofactor.
Interestingly, bundle formation occurs in the absence of the
cofactor. This is in contrast to the previous work with (DPP)FeIII,
where the (porphinato)iron unit played a prominent role in the
formation of the bundle as shown by enhanced helical formation
in the presence of the cofactor.10,12 CD spectra of AHis, BThr
,
and AHis:BThr are consistent well folded R-helical bundles. In
addition, size exclusion chromatography suggests partial forma-
tion of multimeric bundles of AHis, BThr, and tetrameric bundles
of AHis:BThr. Where AUC of the peptides alone suggest bundle
formations of a trimeric AHis, a tetrameric BThr, and a mixture
of oligomeric states exists when both AHis and BThr peptides
are present. These data are consistent with the fact that the trimer
forming AHis peptide is underpacked due to the lack of interior
hydrophobic groups. The BThr peptide has bulky Phe groups
that complement the AHis peptide. In the presence of the
(DPP)Zn chromophore, the preformed bundles/oligomeric states
reorganize to form the targeted heterotetrameric bundle.
We have shown here through computational design and
experimental characterization, a high level of control over the
formation of complex metalloporphyrin-peptide bundles. The
primary coordination sphere is illustrated in electronic absorption
spectroscopy experiments which highlight a red-shift of the
(DPP)Zn B- and Q-bands with binding of a nitrogenous base
(histidine) to the cofactor’s axial position. Circular dichroism
spectroscopic data highlight the secondary structure of the
bundle; AHis, BThr, and AHis:BThr, as well as the complexed
peptide AHis:BThr:(DPP)Zn form well-folded, stable R-helices.
Tertiary structure and predicted bundle formation were con-
firmed by spectrophotometric titrations and analytical ultracen-
Conclusion
In summary, a de noVo light-emissive protein has been
computationally designed and engineered. The protein organizes
two synthetic PZn chromophores in a targeted spatial arrange-
ment and molecular environment. The binding site of this protein
is selective for both metal ion and the nature of the peripheral
porphyrin macrocycle substituents. The protein achieves a
sophisticated level of cofactor discrimination: the heterotet-
rameric, C2-symmetric bundle, AHis:BThr, uniquely binds the
abiological (DPP)Zn chromophore with only a single histidine
coordination. The immeasurably low binding affinity of this
protein for analogous (DPP)Fe cofactors or for zinc hemin
derivatives [e.g., (PPIX)Zn] confirms the utility of probabilistic
computational design77,78 in arriving at proteins complementary
to a chosen cofactor and lays the groundwork for the design of
proteins that incorporate more complex light-addressable elements.
Acknowledgment. This work was supported by the National
Institutes of Health (R01 GM-071628). We gratefully acknowledge
a grant from the U.S. Department of Energy Biomolecular Materials
Program, DE-FG02-04ER46156, for supporting the protein design
elements of this study. We thank the MRSEC (DMR-00-79909)
and NSEC (DMR-0425780) programs of the National Science
Foundation for infrastructural support. M.J.T. is grateful to the
Francqui Foundation (Belgium), and VLAC (Vlaams Academisch
Centrum), Centre for Advanced Studies of the Royal Flemish
Academy of Belgium for Science and the Arts, for research
fellowships.
trifugation. The correct stoichiometry of 1:1:1 AHis:BThr
:
(DPP)Zn was clearly evinced in the titration data, and tetrameric
bundle formation was supported by AUC experiments.
Controlled Organization of Cofactors. Directed-assembly of
multiple chromophores in a single protein via computational
design is necessary when protein function requires vectorially
organized pigments. Such control is manifest in biological light-
harvesting and energy-transducing proteins; a similar ability to
structurally organize light-addressable cofactors is required in
order to fabricate de noVo proteins that possess opto-electronic
functionality. The positive exciton coupling evident in CD
spectra is consistent with the positioning of the chromophores
in proximity to one another. This ability to organize (porphi-
nato)zinc units and maintain important photophysical properties
in the protein bundle such as a nanosecond singlet excited-state
lifetime and a fluorescence rate constant that resembles that
determined in organic solvents (kF ) 4 × 108 s-1), confirms de
noVo protein design as a useful approach to organize emissive
moieties in a unidirectional manner (i.e., along the superhelical
axis of the designed bundle), without alteration of chromophore
properties.
Supporting Information Available: Compiled benchmark
(porphinato)zinc X-ray crystallographic data, detailed binding
model, size exclusion chromatography, additional absorption and
emission spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA907407M
(77) Kang, S. G.; Saven, J. G. Curr. Opin. Chem. Biol. 2007, 11, 329–
334.
(78) Saven, J. G. Curr. Opin. Colloid Interface Sci. 2010, 15, 13-17.
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