Structural Characterisation of the Human α-Lactalbumin Molten Globule at High Temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N–1H HSQC NMR spectrum of the human α-lactalbumin (α-LA) molten globule at pH 2 and 20 °C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 °C leads to a dramatic sharpening of peaks in the 15N–1H HSQC spectrum of human α-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human α-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the Hα, HN and 15N chemical shifts of the human α-LA molten globule at 50 °C leads to the identification of regions of native-like helix in the α-domain and of non-native helical propensity in the β-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.