Self-assembly of peptide porphyrin complexes: toward the development of smart biomaterials.
ABSTRACT The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.
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ABSTRACT: AQ-Pal14 is a 30-residue polypeptide that was designed to form an α-helical coiled coil that contains a metal-binding 4-pyridylalanine residue on its solvent-exposed surface. However, characterization of this peptide shows that it exists as a three-stranded coiled coil, not a two-stranded one as predicted from its design. Reaction with cobalt(III) protoporphyrin IX (Co-PPIX) produces a six-coordinate Co-PPIX(AQ-Pal14)(2) species that creates two coiled-coil oligomerization domains coordinated to opposite faces of the porphyrin ring. It is found that this species undergoes a buffer-dependent self-assembly process: nanometer-scale globular materials were formed when these components were reacted in unbuffered H(2)O, while millimeter-scale, rod-like materials were prepared when the reaction was performed in phosphate buffer (20 mM, pH 7). It is suggested that assembly of the globular material is dictated by the conformational properties of the coiled-coil forming AQ-Pal14 peptide, whereas that of the rod-like material involves interactions between Co-PPIX and phosphate ion.Biomacromolecules 10/2010; 11(10):2602-9. · 5.48 Impact Factor
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ABSTRACT: Helical bundles which bind heme and porphyrin cofactors have been popular targets for cofactor-containing de novo protein design. By analyzing a highly nonredundant subset of the protein databank we have determined a rotamer distribution for helical histidines bound to heme cofactors. Analysis of the entire nonredundant database for helical sequence preferences near the ligand histidine demonstrated little preference for amino acid side chain identity, size, or charge. Analysis of the database subdivided by ligand histidine rotamer, however, reveals strong preferences in each case, and computational modeling illuminates the structural basis for some of these findings. The majority of the rotamer distribution matches that predicted by molecular simulation of a single porphyrin-bound histidine residue placed in the center of an all-alanine helix, and the deviations explain two prominent features of natural heme protein binding sites: heme distortion in the case of the cytochromes C in the m166 histidine rotamer, and a highly prevalent glycine residue in the t73 histidine rotamer. These preferences permit derivation of helical consensus sequence templates which predict optimal side chain-cofactor packing interactions for each rotamer. These findings thus promise to guide future design endeavors not only in the creation of higher affinity heme and porphyrin binding sites, but also in the direction of bound cofactor geometry.Proteins Structure Function and Bioinformatics 02/2009; 74(2):400-16. · 3.39 Impact Factor
Self-Assembly of Peptide Porphyrin Complexes: Toward the Development of
Brian C. Kovaric,†Bashkim Kokona,‡Alexander D. Schwab,†,‡,§Margaret A. Twomey,‡
Julio C. de Paula,†,|and Robert Fairman*,‡
Department of Biology and Department of Chemistry, HaVerford College, 370 Lancaster AVenue,
HaVerford, PennsylVania 19041
Received September 15, 2005; E-mail: email@example.com
Peptides and proteins have been studied as potential biomaterials
because of the strict amino acid sequence control afforded by
synthetic chemical methods and their ability to fold and self-
assemble into well-defined three-dimensional structures, ultimately
leading to nanoscale assemblies with tailor-made structures and
properties.1For example, peptides have been engineered to self-
assemble into nanofilaments with structures that respond to solution
conditions.2Chemical components capable of inducing folding by
tightly binding to peptides can impart novel environmental respon-
siveness and functionalities not typically seen in purely peptidic
materials, expanding the utility of engineered peptide assemblies.
For example, adding porphyrin, or chemically similar moieties, to
a peptide assembly imparts the ability to catalyze and photosensitize
chemical reactions, store oxygen, transport electrical charge, or
transfer molecular excitation energy.3-6
In this study, we demonstrate that an anionic porphyrin, meso-
tetrakis(4-sulfonatophenyl)porphine (TPPS4), induces a coiled-coil
structure in a designed peptide, Cp3K-N, resulting in a tightly bound
porphyrin-peptide pair. The amino acid sequence for Cp3K-N
(Figure 1) is derived from a longer peptide sequence, Cp3K,
designed to self-assemble into one-dimensional coiled-coil nano-
filaments.2Cp3K-N contains three lysines spaced three residues
apart from one another. Isoleucines and leucines spaced three and
four residues apart, respectively, act as determinants for dimeric
coiled-coil motifs.7Solutions containing Cp3K-N and TPPS4in 10
mM Tris-HCl at pH 7.6 were studied with UV-vis spectroscopy
(UV-vis), circular dichroism spectroscopy (CD), and analytical
Evidence for binding of the porphyrin to the peptide, resulting
in induced R-helical content in the peptide, is shown by UV-vis
(Figure 1a) and CD (Figure 1b) measurements, respectively. As
seen in previous studies of porphyrin binding to peptides and
nucleotides, the absorbance of the porphyrin Soret band at 413 nm
decreases with increasing peptide concentration.9-11Unlike many
of these studies, however, a blue-shifted peak appears at 403 nm
upon porphyrin-peptide binding rather than a red-shifted peak, and
the porphyrin Qxbands are red-shifted upon binding (Figure 1a,
inset).9-11These indications of a strong Cp3K-N/TPPS4interaction
are accompanied by significant changes in the Cp3K-N CD spectra
(Figure 1b) which indicate that the largely unfolded structure of
Cp3K-N in the absence of TPPS4is converted into an R-helical
structure with increasing stoichiometric ratio of TPPS4to Cp3K-
N. At a 1:1 stoichiometry, the peptide is largely in a helical
conformation (∼82%) as judged by the ellipticity of the band at
222 nm and the spectral shift of the higher-energy band to 208
nm.12The porphyrin Soret band (Figure 1b, inset) also shows CD,
indicating the existence of a specific porphyrin binding site on the
helical peptide involving perhaps close porphyrin-porphyrin
Titrimetric experiments (Figure 2) illustrate the strength and
stoichiometry of the Cp3K-N/TPPS4 interaction. Excesses of
Cp3K-N or TPPS4beyond the stoichiometry of 1:1 produce no
further decrease in Soret band absorbance or increase in peptide
R-helical content. Given the apparent two-state behavior observed
in the UV-vis experiment (as judged by the isosbestic point), we
treat the data using a simple binding model to obtain a dissociation
constant of 2.09 ( 0.46 µM.8This Kd is comparable to those
measured for peptides specifically engineered to bind porphyrins,
though those peptides lack the readily identifiable structural
transition that accompanies peptide binding seen here.10
Adding 0.15 M NaCl significantly reduces the R-helix content
observed in porphyrin-peptide mixtures, suggesting that binding
has a significant electrostatic component involving interactions
between the porphyrin sulfonates and the peptide lysines (Figure
3). No binding is seen when a cationic porphyrin, meso-tetra(N-
methyl-4-pyridyl)porphine, is used.8The reversible electrostatic
porphyrin attachment seen here, an important paradigm for self-
assembling systems, offers distinct advantages over irreversible
porphyrin-peptide incorporation strategies wherein peptide chains
†Department of Chemistry.
‡Department of Biology.
§Current Address: Department of Chemistry, Appalachian State University,
525 Rivers St., Boone, NC 28608.
|Current Address: Department of Chemistry, Lewis and Clark College, 0615
SW Palatine Hill Rd., Portland, OR 97219.
Figure 1. (a) Series of absorbance spectra taken from solutions containing
various Cp3K-N concentrations and [TPPS4] ) 20 µM (inset: porphyrin
Q-bands). (b) CD spectra from various Cp3K-N/TPPS4 solutions (inset:
porphyrin Soret band). The amino acid sequence of Cp3K-N is Ac-
Published on Web 03/14/2006
4166 9 J. AM. CHEM. SOC. 2006, 128, 4166-4167
10.1021/ja056357q CCC: $33.50 © 2006 American Chemical Society
have been covalently attached to porphyrins or ligated to the metal
centers of metalloporphyrins;4a,13such work has been reviewed
extensively.14For example, the electrostatic mode of binding allows
incorporation of various metals into the porphyrin centers making
the porphyrin-peptide complexes useful as catalysts, photo-
sensitizers, or metal scavenging materials.3
Longer peptide sequences (i.e. Cp3K) are known to dimerize,
forming a coiled-coil.2,7The ability of shorter sequences derived
from Cp3K (i.e., Cp3K-N), induced to form an R-helix by TPPS4,
to form higher order assemblies was determined using AU
sedimentation equilibrium measurements. When prepared individu-
ally, both Cp3K-N and TPPS4 sedimented at their covalent
molecular weights. Global analysis of data sets collected at three
speeds for a porphyrin-peptide mixture revealed an apparent
molecular weight of 6710 ( 330 Da. This suggests higher-order
oligomerization, assuming a 1:1 complex stoichiometry with a
theoretical molecular weight of 2696 Da. The square roots of
variance obtained by numerically fitting AU data using several
theoretical models are shown in Table 1.8
Consistent with the speed dependence of the apparent molecular
weight, an indication of polydispersity, two-species models fit the
data better than any single-species model.8The data are consistent
with dimeric coiled coils decorated with two porphyrins that weakly
associate most likely through porphyrin-porphyrin interactions. The
ability of TPPS4to specifically bind and induce the assembly of a
coiled coil offers the promise of creating responsive materials that
are electronically and photonically active. Our previous work on
longer peptides that form one-dimensional micron-sized polymers
suggests that, with the incorporation of porphyrin derivatives, we
can form long porphyrinic-peptide arrays capable of electron or
excitation energy transfer.2,5,6
Kovaric, who passed away tragically in 2005. We acknowledge
grants from NSF (MCB-0211754 to R.F.), the David and Lucile
Packard Foundation, and the HHMI Undergraduate Science Educa-
tion Program. We thank K. Akerfeldt, S. Amador-Kane, W. Smith,
R. Manning, and K. Johnson for helpful discussions.
This manuscript is dedicated to Brian
Supporting Information Available: Experimental information
available as pdf file. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Figure 2. Dependence of absorbance (at 413 nm) and ellipticity (at 222
nm) on stoichiometric ratio from two titration experiments. In the absorbance
measurements, [TPPS4] was fixed at 50 µM and [Cp3K-N] was varied,
whereas, in the CD measurements, [Cp3K-N] was fixed at 100 µM and
[TPPS4] was varied. Lines have been added as a guide to the eye.
Figure 3. Model of a 1:1 complex of TPPS4/Cp3K-N. The closest distances
between the anionic sulfonate groups and the cationic lysines are labeled.7
Table 1. Square Roots of Variances (SQOV) Calculated by Fitting
AU Sedimentation Equilibrium Data to Various Solution Models
one-state modelsequilibrium models
ratioSQOV (×10-3)ratio SQOV (×10-3)
aValue does not significantly change if a 1:1-2:2-4:4 model is used.
C O M M U N I C A T I O N S
J. AM. CHEM. SOC. 9 VOL. 128, NO. 13, 2006 4167