Supporting Material From Static Structure to Living Protein: Computational Analysis of Cytochrome c Oxidase Main-chain Flexibility

ProFlex calculates a hydrogen-bond dilution profile (1). To calculate the profile, the protein’s hydrogen bonds are broken one by one, from weakest to strongest (according to the potential energy formula described below), and the constraint counting algorithm is run after each bond is broken. This simulates incremental thermal denaturation of the structure, as the calculated temperature rises and hydrogen bonds weaker than the current energy level are broken. The protein commonly appears as a single large rigid region when the simulated temperature is low and weak hydrogen bonds and salt bridges are included. The structure then gradually breaks into two or more rigid regions (often corresponding to the known native state), before going through a cooperative phase transition to a completely flexible chain as the simulated temperature rises (2). Hydrophobic interactions are maintained throughout the process, as their strength actually increases somewhat with modest increases in temperature (3).