J. MoL Biol. (1995) 246, 382-387 JMB COMMUNICATION An Equilibrium Partially Folded State of Human Lysozyme at Low pH Petra Haezebrouck Marcel Joniau 1, Herman Van Dael Shaun D. Hooke 2, Nicholas D, Woodruff 2 and Christopher M. Dobson 2 'Interdisciplinary Research Centre, K. U. Leuven Campus Kortrijk, B-8500 Kortrijk Belgium 2Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road Oxford, OX1 3QT, U.K. *Corresponding author Temperature-induced unfolding of human lysozyme has been monitored by circular dichroism and by nuclear magnetic resonance experiments at a variety of low pH values. The results indicate that, although at pH values above 3 unfolding appears to be consistent with a two-state model, at lower pH values this is not the case. At pH 1.2, for example, unfolding of the tertiary structure occurs at a temperature approximately 10 deg. C lower than that of the secondary structure. At 60°C there is no detectable native tertiary structure remaining for human lysozyme at pH 1.2, although far-UV CD results show preservation of some 40% of the signal attributable to o~-helical elements in the protein. This indicates the existence of a partially folded state of human lysozyme at low pH that has at least some characteristics of the well-defined molten globule state of the homologous cMactalbumins and of the kinetic intermediates observed in the folding of cMactalbumins and of c-type lysozymes. These results suggest that the absolute distinction between these two groups of proteins in terms of their different unfolding behaviour is not valid, and provide insights into possible features stabilizing such states. Keyzoords: protein folding; human lysozyme; molten globule; circular dichroism; intermediate state It is considered an established fact that the denaturation of c-type lysozymes occurs without any thermodynamically stable intermediate forms (Tanford, 1970; Khechinashvili et al., 1973). The thermodynamics and folding behaviour of l);sozymes from several species under equilibrium conditions have been found to accord well with a cooperative two-state model (Radford et al., 1992). Even at low pH, where the thermal transition temperature drops from about 78°C (pH 4.5) to 45°C (pH 1), differential scanning calorimetry (DSC) thermograms of hen egg-white lysozyme are characteristic of cooperative unfolding (Cooper et al., 1992). Another calorimetric stud~ in this case of human lysozyme, again shows that the unfolding of this protein in the pH range from 1.8 to 4.5 occurs in a well-defined two-state process (Herning et al., 1992). This behaviour of lysozymes has been contrasted to that of the homologous c~-lactalbumins, which form stable equilibrium unfolding intermediates Abbreviations used: DSC, differential scanning calorimetry; CD, circular dichroism; t.,, midpoint temperature of thermal denaturation. when placed under mildly denaturing conditions (Dolgikh et al., 1981; Ikeguchi et al., 1986a). The partially folded state of (~-lactalbumin, commonly called a molten globule state, has been characterized by a variety of techniques and shown to exhibit a high content of secondary structure and considerable compactness, but to lack specificity in its tertiary interactions and to possess significant structural flexibility (Kuwajima, 1989; Haynie & Freire, 1993). The molten globule states of cMactalbumins formed under acidic conditions (A-states) have been extensively studied by two-dimensional NMR and hydrogen exchange techniques and found to possess a high degree of disorder in the majority of their side-chains, although conformational preferences in some hydrophobic clusters are evident (Baum et al., 1989; Alexandrescu et al., 1993). That persistent secondary structure exists is, however, revealed by the fact that a number of main-chain amide groups of helical residues that are strongly protected in the native state are also protected in the A-state (Baum et al., 1989; Chyan et al., 1993). The equilibrium molten globule of c~-lactalbumin has been shown to be similar to or identical with a kinetic intermediate transiently accumulated at an 0022-2836/95/080382-06 $08.00/0 © 1995 Academic Press Limited