VIBRATIONAL RAMAN OPTICAL-ACTIVITY OF ALPHA-LACTALBUMIN - COMPARISON WITH LYSOZYME, AND EVIDENCE FOR NATIVE TERTIARY FOLDS IN MOLTEN GLOBULE STATES

Proteins in aqueous solution are now accessible to Raman optical activ ity (ROA) measurements, which provide an incisive new probe of seconda ry and tertiary structure illustrated here by a study of bovine alpha- lactalbumin. The room-temperature ROA spectrum of native bovine alpha- lactalbumin is similar to that of native hen egg-white lysozyme except for features attributable to differences in the loop regions: in part icular, a positive ROA band at similar to 1338 cm(-1) assigned to conf ormationally homogeneous loop structure, possibly with local order cor responding to 3(10)-helix, has more than double the intensity in alpha -lactalbumin compared with lysozyme. This is consistent with the two p roteins having similar secondary structure but different local details in the tertiary fold. ROA measurements on alpha-lactalbumin at pH 2.0 over a range of temperatures have provided a new perspective on the m olten globule state. Thus at 35 degrees C ROA reveals the presence of some secondary structure but an almost complete loss of the tertiary l oop structure; whereas at 2 degrees C the ROA spectrum is almost ident ical with that of the native protein, which is strong evidence that vi rtually all of the secondary structure and the tertiary backbone fold persist, albeit within a looser framework associated with increased so lvent 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 si gnals together with aromatic fluorescence and near-UV circular dichroi sm signals characteristic of the molten globule state. Our sample of a cid alpha-lactalbumin at 2 degrees C therefore appears to be an archet ypal example of Ptitsyn's ''native-like'' molten globule, having a fix ed native-like tertiary fold but with loss of tight packing of the sid e-chains; whereas at 35 degrees C it is a ''disordered'' molten globul e. At 20 degrees C the acid molten globule appears to retain highly na tive-like secondary structure but with most of the tertiary fold alrea dy lost. A calcium-free sample of alpha-lactalbumin at neutral pH disp layed a broad cooperative transition between native and molten globule states at similar to 15 degrees C, with the latter state showing simi lar but somewhat degraded tertiary loop ROA signatures to the native p rotein. In both the acid and apo molten globule states the ROA signatu res of the secondary structure and the tertiary loops showed a gradual change with temperature. (C) 1995 Academic Press Limited