Domain-domain interactions in high mobility group 1 protein (HMG1)

The high mobility group protein HMG1 is a conserved chromosomal protein wit h two homologous DNA-binding domains, A and B, and an acidic carboxy-termin al tail, C. The structure of isolated domains A and B has been previously d etermined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetr y and circular dichroism we have investigated the thermal stability of HMG1 , of the truncated protein A-B (HMG1 without the acidic tail C) and of the isolated domains A and B. In 3 mM sodium acetate buffer, pH 5, the thermal melting of domains A and B are identical (transition temperature t(m) = 43 degrees C and 41 degrees C, denaturation enthalpies Delta H = 46 kcal.mol(- 1)). The thermal melting of protein A-B presents two nearly identical trans itions (t(m) = 40 degrees C and 41 degrees C, Delta H = 44 kcal mol(-1) and 46 kcal.mol(-1), respectively). We conclude that the two domains A and B w ithin protein A-B behave as independent domains. The thermal melting of HMG 1 is biphasic. The two transitions have a different value of t(m) (38 degre es C and 55 degrees C) and corresponding values of Delta H around 40 kcal.m ol(-1). We conclude that within HMG1, the acidic tail C is interacting with one of the two domains A and B, however, the two domains A and B do not in teract with each other. At 37 degrees C, one of the two domains A and B, wi thin HMG1, is partly unfolded, whereas the other which interacts with the a cidic tail C, is fully native. The interaction free energy of the acidic ta il C is estimated to be in the range of 2.5 kcal.mol(-1) based on simulatio ns of the thermograms of HMG1 as a function of the interaction free energy.