Design and characterization of asparagine- and lysine-containing alanine-based helical peptides that bind selectively to A.T base pairs of oligonucleotides immobilized on a 27 mhz quartz crystal microbalance.
We have systematically designed and synthesized six kinds of 16-17 mer alanine-based peptides containing four to six lysine (K) and one to four asparagine (N) residues to achieve the selective binding to A.T base pairs of DNA duplexes. The position and number of K and N residues were changed in the helical structure according to common features of the DNA-binding proteins, in which K and N residues are expected to interact electrostatically with phosphate groups and to interact with A.T base pairs by hydrogen bonding, respectively. The time courses of binding of these peptides to dA(30).dT(30) and dG(30).dC(30) duplexes immobilized on a 27 MHz quartz crystal microbalance (QCM) were studied in 10 mM phosphate buffer (pH 7.5) and 40 mM NaCl at 10 degrees C. The maximum binding amounts (Deltam(max)) on a nanogram scale and binding constants (K(a)) could be obtained from the frequency decrease (mass increase) of the oligonucleotide-immobilized QCM. The conformation changes of the peptides upon binding to DNAs were monitored by circular dichroism (CD) spectroscopy. The four properly arranged N residues in the six-cationic K peptide, K6N4(d), resulted in a 5-fold higher affinity for A.T base pairs (K(a) = 5.9 x 10(5) M(-1)) than for G.C base pairs (K(a) = 1.2 x 10(5) M(-1)), and alpha-helices were clearly promoted by the binding to A.T base pairs from CD spectral changes.
Matsuno
H
H
Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Niikura
K
K
Okahata
Y
Y
D000225:Adenine / Q000737:chemistry; D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D020029:Base Pairing; D015374:Biosensing Techniques / Q000295:instrumentation; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000138:chemical synthesis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013941:Thymine / Q000737:chemistry
D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D010455:Peptides; D011089:Polydeoxyribonucleotides; D001216:Asparagine; D000225:Adenine; D008239:Lysine; D000409:Alanine; D013941:Thymine