Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns.
A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.
Usui
Kenji
K
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama 226-8501, Japan.
Takahashi
Mizuki
M
Nokihara
Kiyoshi
K
Mihara
Hisakazu
H
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D007202:Indicators and Reagents; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D040081:Protein Array Analysis; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007202:Indicators and Reagents; D010455:Peptides