Characterization of millisecond time-scale dynamics in the molten globule state of α-lactalbumin by NMR

Abstract

The motional dynamics of the molten globule (MG) state of α-lactalbumin have been characterized using 15N transverse relaxation rates ( R 2). A modified version of the Carr-Purcell-Meiboom-Gill (CPMG) R 2 pulse sequence is proposed in order to overcome the loss of sensitivity that arises from extreme line broadening due to complex dynamics on the millisecond time-scale. Using this pulse sequence, chemical exchange rates were extracted by examining the 15N transverse relaxation rates as a function of CPMG delay values. The results clearly illustrate that pervasive conformational exchange of 0.2-0.5 ms in the 15N backbone resonances of the molten globule state of α-lactalbumin. The temperature dependence of the conformational exchange rates display standard Arrhenius kinetic behavior between 10 and 30 °C. Estimates of the activation energies range from 0.8 to 4.4 kcal/mol, indicating a low energetic barrier to conformational fluctuations relative to native state proteins. The fluctuations and low energetic barriers may be critical for directing the search for contacts that will result in the transition from the MG state to the native state.