Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer.
High-mobility-group proteins HMG-1 and HMG-I/Y bind to multiple sites within a 268 bp A/T-rich enhancer element of the pea plastocyanin gene ( PetE ). Within a 31 bp region of the enhancer, the binding site for HMG-1 overlaps with the binding site for HMG-I/Y. The kinetics of binding and the affinities of HMG-1 and HMG-I/Y for the 31 bp DNA were determined using surface plasmon resonance. Due to very high non-specific interactions of the HMG proteins with a carboxymethyl-dextran matrix, a novel method using a cholesterol tag to anchor the DNA in a supported lipid monolayer on a thin gold film was devised. The phosphatidylcholine monolayer produced a surface that reduced background interactions to a minimum and permitted the measurement of highly reproducible protein-DNA interactions. The association rate constant ( k (a)) of HMG-I/Y with the 31 bp DNA was approximately 5-fold higher than the rate constant for HMG-1, whereas the dissociation constant ( K (D)) for HMG-I/Y (3.1 nM) was approximately 7-fold lower than that for HMG-1 (20.1 nM). This suggests that HMG-I/Y should bind preferentially at the overlapping binding site within this region of the PetE enhancer.
Webster
C I
CI
Cambridge Centre for Molecular Recognition, University of Cambridge, UK.
Cooper
M A
MA
Packman
L C
LC
Williams
D H
DH
Gray
J C
JC
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002784:Cholesterol / Q000737:chemistry; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D017343:Genes, Plant; D025741:HMGA1a Protein; D006609:High Mobility Group Proteins / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D010970:Plastocyanin / Q000235:genetics; D011485:Protein Binding; D020349:Surface Plasmon Resonance
D018744:DNA, Plant; C107092:HMGA1a Protein, plant; D006609:High Mobility Group Proteins; D008055:Lipids; D010940:Plant Proteins; D025741:HMGA1a Protein; D010970:Plastocyanin; D002784:Cholesterol