The high mobility group protein HMG1 is a conserved chromosomal protein with two homologous DNA-binding domains, A and B, and an acidic carboxy-terminal tail, C. The structure of isolated domains A and B has been previously determined by NMR, but the interactions of the different domains within the complete protein were unknown. By means of differential scanning calorimetry 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 transitions (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 within protein A-B behave as independent domains. The thermal melting of HMG1 is biphasic. The two transitions have a different value of t(m) (38 degrees C and 55 degrees C) and corresponding values of Delta H around 40 kcal.mol(-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 interact with each other. At 37 degrees C, one of the two domains A and B, within HMG1, is partly unfolded, whereas the other which interacts with the acidic tail C, is fully native. The interaction free energy of the acidic tail C is estimated to be in the range of 2.5 kcal.mol(-1) based on simulations of the thermograms of HMG1 as a function of the interaction free energy.
File in questo prodotto:
Non ci sono file associati a questo prodotto.