Difference in Affinity for DNA between HMG Proteins 1 and 2 Determined by Surface Plasmon Resonance Measurements

Abstract

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.

<title>Probing protein: DNA interactions using a uniform monolayer of DNA and surface plasmon resonance</title>

“…The use of a biotin-SA linker for immobilization of oligonucleotides for studies of protein:DNA binding using columns and gels has been well proven in a number of experiments to allow the immobilized dsDNAs to remain active and selective in their protein binding [7,[12][13][14][15][16][17][18][19][20][21][22][23]. To our knowledge this is the first demonstration d the use of a biotin-SA linker for immobilization of oligonucleotides to study protein:DNA binding with a planar dsDNA monolayer.…”

SRY and Human Sex Determination: The Basic Tail of the HMG Box Functions as a Kinetic Clamp to Augment DNA Bending

“…10 K2 s K1 (k off ). 72 Stopped-flow kinetic studies of the binding of the HMG-1A domain to cis-platinmodified DNA showed that the association rate is near the diffusion limit. Similarly, dissociation rates of cis-platin-modified DNA complexes are significantly faster than those of the unmodified DNA complexes.…”

“…As in LEF-1, [45][46][47][48] the SRY tail augments specific DNA binding and bending, but in SRY these effects are modest: DNA binding is reduced by less than twofold and DNA bending by only 7-108. Further, binding of the tail does not damp long-range fluctuations in DNA 42 Ribbon model of protein is shown in green (HMG box) and red (tail; residues [70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]. DNA is shown as gray sticks.…”

“…(b) LEF1-DNA complex (PDB accession code 2LEF). 37 A ribbon model of the protein is shown in blue (HMG box) and red (tail; residues [70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85]. DNA is shown as gray sticks.…”

Interaction of hnRNP A2/B1 Isoforms with Telomeric ssDNA and the in Vitro Function

“…Three biotinylated oligomers, (TTAGGG) 6 , (TTAGGG) 3 , and TTAGGG, were immobilized in separate flow channels of a sensor chip (SA, AmershamPharmacia Biotech). The in vitro interaction with purified B1, B0b and single-strand binding proteins (SSBs) of E. coli (Pharmacia Biotech) were monitored and analyzed using a ver2.1 BIAevaluation software to calculate dissociation and association rate constants (17). The electrophoretic mobility shift assay was also performed to determine the dissociation constants.…”