Kinetic analysis of acetylation-dependent Pb1 bromodomain-histone interactions.
Stopped-flow fluorescence anisotropy was used to determine the kinetic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains are acetyllysine binding motifs found in many chromatin associated proteins. Individual bromodomains were derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin-remodeling complex that has six tandem bromodomains in the amino-terminal region. The average k(on) and k(off) values for the formation of high-affinity complexes are 275 M(-1) s(-1) and 0.41 x 10(-3) s(-1), respectively. The average k(on) and k(off) values for the formation of low-affinity complexes are 119 M(-1) s(-1) and 1.42 x 10(-3) s(-1), respectively. Analysis of the on- and off-rates yields acetylation site-dependent equilibrium dissociation constants averaging 1.4 and 12.9 microM for high- and low-affinity complexes, respectively. This work represents the first examination of kinetic mechanisms of acetylation-dependent bromodomain-histone interactions.
Department of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, United States.
Christopher
C
Kupitz
Renu
R
Chandrasekaran
Martin
M
Thompson
D000107:Acetylation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins; D005454:Fluorescence Polarization; D006657:Histones / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry*
D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; C102195:PBRM1 protein, human; D010455:Peptides; D014157:Transcription Factors