Vibrational Raman optical activity of α-lactalbumin: comparison with lysozyme, and evidence for native tertiary folds in molten globule states

Proteins in aqueous solution are now accessible to Raman optical activity (ROA) measurements, which provide an incisive new probe of secondary and tertiary structure illustrated here by a study of bovine α-lactalbumin. The room-temperature ROA spectrum of native bovine α-lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in particular, a positive ROA band at ∼1338 cm−1assigned to conformationally homogeneous loop structure, possibly with local order corresponding to 310-helix, has more than double the intensity in α-lactalbumin compared with lysozyme. This is consistent with the two proteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on α-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the molten globule state. Thus at 35°C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary loop structure; whereas at 2°C the ROA spectrum is almost identical with that of the native protein, which is strong evidence that virtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased solvent exposure and change of environment of many of the side-chains as evidenced by an increase in noise and bandwidth of some of the ROA signals together with aromatic fluorescence and near-UV circular dichroism signals characteristic of the molten globule state. Our sample of acid α-lactalbumin at 2°C therefore appears to be an archetypal example of Ptitsyn's "native-like" molten globule, having a fixed native-like tertiary fold but with loss of tight packing of the side-chains; whereas at 35°C it is a "disordered" molten globule. At 20°C the acid molten globule appears to retain highly native-like secondary structure but with most of the tertiary fold already lost. A calcium-free sample of α-lactalbumin at neutral pH displayed a broad cooperative transition between native and molten globule states at ∼15 °C, with the latter state showing similar but somewhat degraded tertiary loop ROA signatures to the native protein. In both the acid and apo molten globule states the ROA signatures of the secondary structure and the tertiary loops showed a gradual change with temperature