Time- and temperature dependence of large-scale conformational transitions in myoglobin

Abstract

Structure, dynamics, and function of proteins are strongly interrelated. We study the structure-function relationship in carbonmonoxy-myoglobin (MbCO) using two techniques: (i) flash photolysis with rebinding monitored in the CO stretch bands over wide ranges in time (≈ 3 μs to 1 s) and temperature (60 to 260 K); (ii) pressure-jump experiments with protein relaxations monitored in the CO stretch bands by FTIR spectroscopy for times between 10 s and 30 ks and temperatures between 155 and 220 K. The three CO stretch bands correspond to three major conformational substates, A 0, A 1, and A 3, with different structures and rebinding kinetics. The flash-photolysis experiments show that the absorbance change of A 0 is nonmonotonic in time during rebinding above about 220 K owing to interconversion of A 0 with A 1 and A 3. The P-jump experiments establish that MbCO experiences large-scale motions which are nonexponential in time, non-Arrhenius in temperature, and strongly dependent on solvent viscosity. One of the motions observed in the P-jump experiments corresponds to the large-scale structural transition, A 0→A 1 + A 3, observed during rebinding. These results, together with earlier data, begin to give a detailed picture of the conformational energy landscape of MbCO: it contains many conformational substates which are hierarchically arranged in at least three tiers. Motions in each of the three tiers affect the MbCO rebinding kinetics.