Structural changes induced by binding of the high-mobility group I proteinto a mouse satellite DNA sequence

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA com plexes formed, as determined by Job plots derived from experiments using py rene-labeled duplexes. Circular dichroism and melting temperature experimen ts extended in the far ultraviolet range show that while native HMG-I is ma inly random coiled in solution, it adopts a beta-turn conformation upon for ming a 1:1 complex in which the protein first binds to one of two dA.dT str etches present in the duplex. HMG-I structure in the 1:1 complex is depende nt on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or co mpaction coupled to a change in the protein conformation, as determined fro m fluorescence resonance energy transfer (FRET) experiments. in addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces a n ordered condensation of DNA duplexes. Based on the constraints derived fr om pyrene excimer measurements, we present a model of these nucleated struc tures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the D NA-binding motifs of HMG-I. We discuss the functional relevance of the stru ctural flexibility of HMG-I associated with the nature of its DNA targets a nd the implications of the binding stoichiometry for several aspects of chr omatin structure and gene regulation.