HMGB1 (high mobility group box protein 1) is an architectural protein that facilitates formation of protein-DNA assemblies involved in transcription, recombination, DNA repair, and chromatin remodeling. Important to its function is the ability of HMGB1 to bend DNA non-sequence specifically. HMGB1 contains two HMG boxes that bind and bend DNA (the A box and the B box) and a C-terminal acidic tail. We investigated how these domains contribute to DNA bending by HMGB1 using single molecule FRET, which enabled us to resolve heterogeneous populations of bent and unbent DNA. We found that full length HMGB1 bent DNA more than the individual A and B boxes. Removing the C-terminal tail resulted in a protein that bent DNA to a greater extent than the full length protein. These data suggest that the A and B boxes simultaneously bind DNA in the absence of the C-terminal tail, but the tail modulates DNA binding and bending by one of the HMG boxes in the full length protein. Indeed, a construct composed of the B box and the C-terminal tail only bent DNA at higher protein concentrations. Moreover, in the context of the full length protein, mutating the A box such that it could not bend DNA resulted in a protein that bent DNA similarly to a single HMG box and only at higher protein concentrations. We propose a model in which the HMGB1 C-terminal tail serves as an intramolecular damper that modulates the interaction of the B box with DNA.

FRET (Forster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students measured the apparent FRET between donor and acceptor fluorophores located on the ends of several DNAs of unknown lengths, enabling them to order the DNAs according to size. In addition, students investigated site-specific DNA cleavage by restriction endonucleases, using loss of apparent FRET to determine which enzyme cut sites were present in each of the DNAs. After completing this experiment, students understood the inverse relationship between changes in FRET and changes in distance, and understood how changes in FRET could be used to monitor a conformational change in a molecule. As an extension to the experiment, a tutorial is included that uses the same DNAs to illustrate the ability of singlemolecule FRET measurements to resolve heterogeneity in a sample, which cannot be done via more traditional ensemble measurements.