Exploration of Partially Unfolded States of Human a-Lactalbumin by Molecular Dynamics Simulation Emanuele Paci 1,2 , Lorna J. Smith 1 , Christopher M. Dobson 1 and Martin Karplus 1,2,3 * 1 Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road Oxford OX1 3QT, UK 2 Laboratoire de Chimie Biophysique, ISIS, Universite  Louis Pasteur, 4 rue Blaise Pascal, 67000 Strasbourg France 3 Department of Chemistry and Biological Chemistry, Harvard University, 12 Oxford Street Cambridge, MA 02138, USA Molecular dynamics simulations are used to probe the properties of non- native states of the protein human a-lactalbumin (human a-LA) with a detailed atomistic model in an implicit aqueous solvent environment. To sample the conformational space, a biasing force is introduced that increases the radius of gyration relative to the native state and generates a large number of low-energy conformers that differ in terms of their root-mean-square deviation, for a given radius of gyration. The resulting structures are relaxed by unbiased simulations and used as models of the molten globule and partly denatured states of human a-LA, based on measured radii of gyration obtained from nuclear magnetic resonance experiments. The ensembles of structures agree in their overall properties with experimental data available for the human a-LA molten globule and its more denatured states. In particular, the simulation results show that the native-like fold of the a-domain is preserved in the molten globule. Further, a considerable proportion of the antiparallel b-strand in the b-domain are present. This indicates that the lack of hydrogen exchange protection found experimentally for the b-domain is due to rearrange- ment of the b-sheet involving transient populations of non-native b-struc- tures. The simulations also provide details concerning the ensemble of structures that contribute as the molten globule unfolds and shows, in accord with experimental data, that unfolding is not cooperative; i.e. the various structural elements do not unfold simultaneously. # 2001 Academic Press Keywords: molecular dynamics; protein folding; protein denaturation; human a-lactalbumin; molten globule *Corresponding author Introduction States of proteins that are intermediate between the native state and the fully unfolded state are of great interest. Some of them occur only during the kinetics of the folding process (Roder & Colo Ân, 1997) while others, usually referred to as molten globules (Ptitsyn, 1995), can be stabilized by environmental conditions, such as low pH. There is evidence for some proteins, e.g. apomyoglobin (Barrick & Baldwin, 1993), that the molten globule present under equilibrium conditions resembles kinetic intermediates formed during protein fold- ing (Jennings & Wright, 1993). This is true also of bovine a-lactalbumin (a-LA; see Figure 11), which has been shown to resemble closely a kinetic inter- mediate on the folding pathway (Balbach et al., 1995; Forge et al., 1999). Moreover, there is evi- dence that the molten globule and other non-native states of some proteins are functionally important (Bychkova et al., 1988; Penkett et al., 1998) and that partly denatured species play a role in the tran- sition to amyloid ®brils (Kelly, 1998; Chiti et al., 1999). Svensson et al. (1999) have suggested that the molten globule state of human a-LA is associ- ated with apoptosis in tumor cells. It is of great interest, therefore, to obtain more information about the structural and thermodynamic properties of these non-native species. However, this is dif®- cult because molten globules have not been crystal- lized and their NMR spectra have broad lines. Molten globules cover a much wider region of conformational space than the native state and may undergo non-cooperative transitions between E-mail address of the corresponding author: marci@brel.u-strasbg.fr Abbreviations used: a-LA, a-lactalbumin; BMD, biased molecular dynamics; MG, molten globule; FTIR, Fourier transform infrared. doi:10.1006/jmbi.2000.4337 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 329±347 0022-2836/01/020329±19 $35.00/0 # 2001 Academic Press