Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance.
We report quantitative analysis of peptide-peptide interactions on a 27 MHz quartz crystal microbalance (QCM) in aqueous solution. The KID (kinase-inducible domain) of transcription factor CREB (cyclic AMP response element binding protein) is known to interact with the KIX domain of coactivator CBP (CREB binding protein), facilitated by phosphorylation at Ser-133 of the KID. The KIX domain peptide (86 aa) was immobilized on the QCM gold electrode surface by means of a poly(ethylene glycol) spacer. Binding of the KID peptide (46 aa) to the KIX peptide was detected by frequency decreases (mass increases) of the QCM. Both maximum binding amount (Deltammax) and association constants (Ka) obtained from the QCM measurements increased as a result of phosphorylation of Ser-133 of the KID peptide. The Ka values for KIX peptide to the phosphorylated (pKID) and unphosphorylated KID peptides were (93+/-2) x 10(3) and (5+/-1) x 10(3) M(-1), respectively. This difference was explained by the dissociation rate constant (k(-1)) of the pKID being 20 times smaller than that of the KID, while association rate constants (k1) were independent of phosphorylation.
Matsuno
Hisao
H
Department of Biomolecular Engineering, Tokyo Institute of Technology and CREST, JST, 4259 Nagatsuda, Midori-ku, Yokohama 226-8501, Japan.
Furusawa
Hiroyuki
H
Okahata
Yoshio
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D050882:CREB-Binding Protein / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021281:Quantitative Structure-Activity Relationship; D011791:Quartz
D017362:Cyclic AMP Response Element-Binding Protein; D010455:Peptides; D011791:Quartz; D050882:CREB-Binding Protein