Dual binding modes for an HMG domain from human HMGB2 on DNA.
High mobility group B (HMGB) proteins contain two HMG box domains known to bind without sequence specificity into the DNA minor groove, slightly intercalating between basepairs and producing a strong bend in the DNA backbone. We use optical tweezers to measure the forces required to stretch single DNA molecules. Parameters describing DNA flexibility, including contour length and persistence length, are revealed. In the presence of nanomolar concentrations of isolated HMG box A from HMGB2, DNA shows a decrease in its persistence length, where the protein induces an average DNA bend angle of 114 +/- 21 degrees for 50 mM Na+, and 87 +/- 9 degrees for 100 mM Na+. The DNA contour length increases from 0.341 +/- 0.003 to 0.397 +/- 0.012 nm per basepair, independent of salt concentration. In 50 mM Na+, the protein does not unbind even at high DNA extension, whereas in 100 mM Na+, the protein appears to unbind only below concentrations of 2 nM. These observations support a flexible hinge model for noncooperative HMG binding at low protein concentrations. However, at higher protein concentrations, a cooperative filament mode is observed instead of the hinge binding. This mode may be uniquely characterized by this high-force optical tweezers experiment.
McCauley
Micah
M
Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115, USA.
Hardwidge
Philip R
PR
Maher
L James
LJ
Williams
Mark C
MC
D000818:Animals; D001703:Biophysics / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004926:Escherichia coli / Q000378:metabolism; D024261:HMGB2 Protein / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D015233:Models, Statistical; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000737:chemistry
D024261:HMGB2 Protein; D011108:Polymers; D004247:DNA; D012964:Sodium