The refolding of apo bovine a-lactalbumin has been monitored in real t
ime by NMR spectroscopy following rapid in situ dilution of a chemical
ly denatured state. By examining individual resonances in the time-res
olved NMR spectra, the native state has been shown to emerge in a coop
erative manner from an intermediate formed in the dead-time of the exp
eriments. The kinetics of folding to the native state are closely simi
lar to those observed by stopped-flow fluorescence and near-UV circula
r dichroism. The NMR spectrum of the transient intermediate resembles
closely that of the well characterized stable molten globule state for
med at low pH. The results suggest that NMR can play a key role in des
cribing at an atomic level the structural transitions occurring during
protein folding.