Molecular Dynamics Simulations of Peptide Fragments from Hen Lysozyme: Insight into Non-native Protein Conformations Lorna J. Smith 1 *, Alan E. Mark 2 , Christopher M. Dobson 1 and Wilfred F. van Gunsteren 1,2 1 Oxford Centre for Molecular Sciences and New Chemistry Laboratory, University of Oxford, South Parks Road Oxford OX1 3QR, England 2 Department of Physical Chemistry, Swiss Federal Institute of Technology Zu È rich ETH-Zentrum, 8092 Zu È rich Switzerland Molecular dynamics simulations of four peptides taken from the hen lysozyme sequence have been used to generate models for non-native protein conformations. Comparisons between the different peptides and with experimental data for denatured lysozyme and peptide fragments provides insight into the characteristics of the conformational ensembles populated in these non-native states and the dependence of their struc- tural features on the amino acid sequence. For the denatured conformers populated local contacts dominate in determining the properties observed in the trajectories, all four peptides showing similar characteristics. These include a signi®cant increase in the number of main-chain O(i)± NH(i  2) hydrogen bonds and hydrogen bonds involving side-chain groups, this increase compensating to a large extent for the loss of hydro- gen bonds involved in helical or b-sheet secondary structure in the native fold, and the generation of a population of collapsed states with local clusterings of hydrophobic groups. The hydrophobic clusters enable at least partial burial of many side-chains exposed by the loss of tertiary contacts on denaturation and provide models that may explain the exper- imentally observed protection of amides from hydrogen exchange and the existence of residual secondary structure in non-native species of lysozyme. The results suggest that this approach has an important role to play in aiding the interpretation of experimental data for conformation- ally disordered non-native states of proteins. # 1998 Academic Press Keywords: molecular dynamics; computer simulation; protein folding; NMR spectroscopy; denatured protein *Corresponding author Introduction There is increasing interest in characterising unfolded and partially folded protein confor- mations as it has been recognised that such states can give considerable insight into issues such as protein stability and folding, and the relationship between the sequence and three-dimensional struc- ture of a protein (Dill & Shortle, 1991; Dobson, 1992; Shortle, 1996a; Smith et al., 1996). In addition, non-native states are involved in a range of import- ant biological processes, including the transport of proteins across membranes and protein degra- dation within the cell (Dill & Shortle, 1991; Ptitsyn, 1995). They are also involved in protein aggrega- tion which is of signi®cance with regard to our understanding of amyloid associated diseases such as Alzheimer's and the spongiform encephalophies (Thomas et al., 1995). Recently a number of pro- teins that are unfolded under physiological con- ditions but are biologically active have also been identi®ed (Gast et al., 1995; Kriwacki et al., 1996; Weinreb et al., 1996; Penkett et al., 1997). De®ning the conformational properties of states that are not fully folded is, however, challenging as they are in general ensembles of interconverting conformers. This can complicate both the experimental measurements, particularly in the case of NMR techniques if there is relatively slow interconver- sion between the conformers, and also the E-mail address of the corresponding author: lorna.smith@chemistry.oxford.ac.uk Abbreviations used: MD, molecular dynamics; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; RMSD, root-mean-square difference. Article No. mb981892 J. Mol. Biol. (1998) 280, 703±719 0022 ± 2836/98/290703±17 $30.00/0 # 1998 Academic Press