DIFFERENCE IN AFFINITY FOR DNA BETWEEN HMG PROTEIN-1 AND PROTEIN-2 DETERMINED BY SURFACE-PLASMON RESONANCE MEASUREMENTS

High mobility group (HMG) proteins 1 and 2 contain two similar but non -identical repeats of DNA-binding domains and an acidic C-terminal, Th e proposed functions of HMG proteins 1 and 2 imply a probable differen ce in their DNA-binding abilities, The primary studies by gel retardat ion assay showed that HMG2 has higher affinity than HMG1 for supercoil ed and linear DNA, The DNA-binding of HMG2 appeared strong enough to a llow exchange with HMG1 molecule already bound to DNA, while the DNA-b inding region of HMG1 showed higher affinity than that of HMG2, In ord er to compare more quantitatively the affinities, surface plasmon reso nance (SPR) measurements using a BIAcore instrument were conducted. Th e kinetic data indicated that the K-d for the complex of HMG;2 with DN A is smaller than that of HMG1, in contrast to the situation for the D NA-binding region of these proteins. The sequence between the second D NA-binding domain and the acidic C-terminal of HMG proteins is require d for tight DNA-binding, Also, the acidic C-terminal strongly modulate s the DNA-binding ability of each protein, The usefulness of SPR measu rement for quantitative analysis of affinity and regions involved in D NA-binding under conditions nearly identical to those in solution is d iscussed.