Fast events in protein folding: Relaxation dynamics and structure of the I form of apomyoglobin

The fast relaxation dynamics of the acid destabilized I form of apomyoglobin (pH* 3, 0.15 M NaCl; apoMb-I) following a laser-induced temperature-jump have been probed using time-resolved infrared spectroscopy. Only a fast, single exponential phase is observed (bleach centered at v = 1633 cm^-1 and transient absorbance at 1666 cm^-1) with relaxation times of 38 ns at 30 °C and 36 ns at 57 °C; no additional slow (microsecond) phase is observed as previously found in the native form of apomyoglobin. Folding times of approximately 66 ns are derived from the observed rates based on a simple two-state model. The equilibrium melting of the 1633 cm^-1 component shows noncooperative linear behavior over the temperature range studied (10- 60 °C). The low amide I' frequency, the fast relaxation dynamics, and the noncooperative melting behavior are characteristic of isolated solvated helix. The analysis of the amide-I' band reveals another major component at 1650 cm^-1 assigned to native-like structure stabilized by tertiary contacts involving the AGH core, which does not show dynamic or static melting under our conditions. ApoMb-I has generally been taken to be a 'molten globule' species. The present results indicate a heterogeneous structure consisting of separate regions of native-like unit(s), solvated helices, and disordered coil, excluding a homogeneous molten globule as a model for apoMb-I. From the current studies and other results, a detailed model of the folding of apomyoglobin is presented.