61
neutral pH. We investigated the ability of self-assembling supramolecular catalysts to catalyze oxidative coupling of
2,6- dimethoxyphenol (DMP), a well-characterized reaction that
produces a bright orange product [13, 14]. Several highly reactive
peptides that are capable to efficiently oxidize DMP using dioxygen as a cofactor were identified. Just like in the case of zincbinding supramolecular catalysts of ester hydrolysis, the
oxygen-activating ability strongly correlated with the propensity of
peptides to form a β-sheet structure.
Catalytic efficiency of the self-assembling peptides is impressive
given the simplicity and adaptability of the design. A large number
of short peptides can be screened in a high-throughput fashion.
Additionally, mixing of different peptides prior to fibril formation
provides access to a large number of different arrangements of
functional groups in a single amyloid-like supramolecular assembly.
Feasibility of this idea has been previously demonstrated: mixing
peptides with different residues at position 6 produced fibrils more
active than fibrils assembled from uniform peptides [7]. Simple
variation of side chains in the hydrophilic positions of the peptide
sequence allows for exploration of many different coordination
arrangements that efficiently support the metal ion in all oxidation states required for catalysis [15]. Enzymes selectively recognize their substrates through multiple contacts with the protein’s
side chains. This property could be potentially engineered into
catalytic fibrils.
Here, we provide a detailed protocol for robust preparation
and kinetic characterization of supramolecular peptide assemblies
that bind Cu
2+
and catalyze phenol oxidation. The assay we developed is based on the benchmarked reaction of DMP oxidation,
often used to assess activity of laccase enzymes [16–19]. This assay
provides a simple readout signal, is easy to perform, and is suitable
for high-throughput screening in 96-well plates. We also present
an optimized protocol for the preparation of heteropeptidic assemblies, where peptides with different primary sequences are incorporated into a single fibril.
2 Materials
All solutions need to be prepared using MilliQ water, HPLC grade
organic solvents, and analytical grade reagents.
1. 1 M Hepes-KOH buffer, pH 8: Weigh out 9.53 g of Hepes
free acid in a 50 mL Falcon tube. Add 30 mL of water to dissolve
Hepes-free acid and adjust pH using 5 M KOH, and then add
water to 40 mL (see Note 1).
2. 50 mM CuSO 4 solution in water: Weigh out 0.5 g of
CuSO 4 *5H 2 O and add water to 40 mL. The final concentration
Preparation and Screening of Amyloid Fibrils
neutral pH. We investigated the ability of self-assembling supramolecular catalysts to catalyze oxidative coupling of
2,6- dimethoxyphenol (DMP), a well-characterized reaction that
produces a bright orange product [13, 14]. Several highly reactive
peptides that are capable to efficiently oxidize DMP using dioxygen as a cofactor were identified. Just like in the case of zincbinding supramolecular catalysts of ester hydrolysis, the
oxygen-activating ability strongly correlated with the propensity of
peptides to form a β-sheet structure.
Catalytic efficiency of the self-assembling peptides is impressive
given the simplicity and adaptability of the design. A large number
of short peptides can be screened in a high-throughput fashion.
Additionally, mixing of different peptides prior to fibril formation
provides access to a large number of different arrangements of
functional groups in a single amyloid-like supramolecular assembly.
Feasibility of this idea has been previously demonstrated: mixing
peptides with different residues at position 6 produced fibrils more
active than fibrils assembled from uniform peptides [7]. Simple
variation of side chains in the hydrophilic positions of the peptide
sequence allows for exploration of many different coordination
arrangements that efficiently support the metal ion in all oxidation states required for catalysis [15]. Enzymes selectively recognize their substrates through multiple contacts with the protein’s
side chains. This property could be potentially engineered into
catalytic fibrils.
Here, we provide a detailed protocol for robust preparation
and kinetic characterization of supramolecular peptide assemblies
that bind Cu
2+
and catalyze phenol oxidation. The assay we developed is based on the benchmarked reaction of DMP oxidation,
often used to assess activity of laccase enzymes [16–19]. This assay
provides a simple readout signal, is easy to perform, and is suitable
for high-throughput screening in 96-well plates. We also present
an optimized protocol for the preparation of heteropeptidic assemblies, where peptides with different primary sequences are incorporated into a single fibril.
2 Materials
All solutions need to be prepared using MilliQ water, HPLC grade
organic solvents, and analytical grade reagents.
1. 1 M Hepes-KOH buffer, pH 8: Weigh out 9.53 g of Hepes
free acid in a 50 mL Falcon tube. Add 30 mL of water to dissolve
Hepes-free acid and adjust pH using 5 M KOH, and then add
water to 40 mL (see Note 1).
2. 50 mM CuSO 4 solution in water: Weigh out 0.5 g of
CuSO 4 *5H 2 O and add water to 40 mL. The final concentration
Preparation and Screening of Amyloid Fibrils