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

The motional dynamics of the molten globule (MG) state of alpha-lactalbumin have been characterized using N-15 transverse relaxation rates (R-2). A mo dified version of the Carr-Purcell-Meiboom-Gill (CPMG) R-2 pulse sequence i s proposed in order to overcome the loss of sensitivity that arises from ex treme Line broadening due to complex dynamics on the millisecond time-scale . Using this pulse sequence, chemical exchange rates were extracted by exam ining the N-15 transverse relaxation rates as a function of CPMG delay valu es. The results clearly illustrate that pervasive conformational exchange o f 0.2-0.5 ms in the N-15 backbone resonances of the molten globule state of alpha-lactalbumin. The temperature dependence of the conformational exchan ge rates display standard Arrhenius kinetic behavior between 10 and 30 degr ees 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 t ransition from the MG state to the native state. (C) 1999 Academic Press.