Difference in affinity for DNA between HMG proteins 1 and 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. The proposed functions of HMG proteins 1 and 2 imply a probable difference in their DNA-binding abilities. The primary studies by gel retardation assay showed that HMG2 has higher affinity than HMG1 for supercoiled and linear DNA. The DNA-binding of HMG2 appeared strong enough to allow exchange with HMG1 molecule already bound to DNA, while the DNA-binding region of HMG1 showed higher affinity than that of HMG2. In order to compare more quantitatively the affinities, surface plasmon resonance (SPR) measurements using a BIAcore instrument were conducted. The kinetic data indicated that the Kd for the complex of HMG2 with DNA is smaller than that of HMG1, in contrast to the situation for the DNA-binding region of these proteins. The sequence between the second DNA-binding domain and the acidic C-terminal of HMG proteins is required for tight DNA-binding. Also, the acidic C-terminal strongly modulates the DNA-binding ability of each protein. The usefulness of SPR measurement for quantitative analysis of affinity and regions involved in DNA-binding under conditions nearly identical to those in solution is discussed.
Yamamoto
A
A
Department of Biological Science and Technology, Science University of Tokyo, Chiba.
Ando
Y
Y
Yoshioka
K
K
Saito
K
K
Tanabe
T
T
Shirakawa
H
H
Yoshida
M
M
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins / Q000378:metabolism*; D007700:Kinetics; D013552:Swine
D002352:Carrier Proteins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D004247:DNA