Nucleic Acids Research, 2000, Vol. 28, No. 7 1618-1624
© 2000 Oxford University Press
Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer
Cambridge Centre for Molecular Recognition and 1Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK, 2Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK and 3Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
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 (ka) of HMG-I/Y with the 31 bp DNA was ~5-fold higher than the rate constant for HMG-1, whereas the dissociation constant (KD) for HMG-I/Y (3.1 nM) was ~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.
* To whom correspondence should be addressed. Tel: +44 1223 333925; Fax: +44 1223 333953; Email: jcg2@mole.bio.cam.ac.uk Present address: Carl I. Webster, Cambridge Antibody Technology, The Science Park, Melbourn, Royston SG8 6JJ, UK
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