Role of Hydrophilic and Hydrophobic Contacts in Folding of the Second -Hairpin Fragment of Protein G: Molecular Dynamics Simulation Studies of an All-Atom Model Yaoqi Zhou * and Apichart Linhananta Howard Hughes Medical Institutes Center for Single Molecule Biophysics, Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York ABSTRACT Predicting the folding mechanism of the second -hairpin fragment of the Ig-binding domain B of streptococcal protein G is unexpectedly challenging for simplified reduced models because the models developed so far indicated a different folding mechanism from what was suggested from high-temperature unfolding and equilibrium free- energy surface analysis based on established all- atom empirical force fields in explicit or implicit solvent. This happened despite the use of empirical residue-based interactions, multibody hydrophobic interactions, and inclusions of hydrogen bonding effects in the simplified models. This article employs a recently developed all-atom (except nonpolar hy- drogens) model interacting with simple square-well potentials to fold the peptide fragment by molecular dynamics simulation methods. In this study, 193 out of 200 trajectories are folded at two reduced tem- peratures (3.5 and 3.7) close to the transition tem- perature T*  4.0. Each simulation takes <7 h of CPU time on a Pentium 800-MHz PC. Folding of the new all-atom model is found to be initiated by collapse before the formation of main-chain hydro- gen bonds. This verifies the mechanism proposed from previous all-atom unfolding and equilibrium simulations. The new model further predicts that the collapse is initiated by two nucleation contacts (a hydrophilic contact between D46 and T49 and a hydrophobic contact between Y45 and F52), in agree- ment with recent NMR measurements. The results suggest that atomic packing and native contact interactions play a dominant role in folding mecha- nism. Proteins 2002;47:154 –162. © 2002 Wiley-Liss, Inc. INTRODUCTION The -hairpin motif is one of the basic building blocks of the structures of proteins. Understanding the folding of a -hairpin is the first step toward the understanding of protein folding. The folding of the second -hairpin frag- ment (residues 41–56) of the Ig-binding domain B of streptococcal protein G ( 2 BpG) is of particular interest because it is the only -hairpin whose folding is proposed to be initiated by hydrophobic collapse, 1–5 rather than by the inter-strand hydrogen bonds near the turn region. 6,7 The latter has been found to be the folding mechanism of a number of other -hairpins, such as the 11-residue peptide VVVVDPGVVVV, 8 the first -hairpin of tendamistat, 9 and a three-stranded antiparallel -sheet peptide. 10 The fold- ing mechanisms of these three peptides were examined by all-atom folding simulations of models interacting with well-established empirical force fields (AMBER, 11 CHARMM, 12 or GROMOS 13 ) in an explicit 9 or implicit 8,10 solvent. Because all-atom folding simulation of  2 BpG with an empirical force field is not yet feasible, the proposed mechanism was inferred from an equilibrium free-energy analysis, 2–5 unfolding simulations 1,4 and computational mutation studies. 4 In these studies, various empirical force fields with atomic details, in an explicit or implicit solvent, were employed. The proposed mechanism, how- ever, differs from the folding simulation results of lattice 14 and off-lattice 15 models with a reduced atomic representa- tion, but with sophisticated interaction scheme designed for the same -hairpin. Both folding simulations suggest that folding is initiated at the turn region. Klimov and Thirumalai used an off-lattice model in which each sidechain is represented by a bead and interacts with each other by an empirical residue-dependent contact energy and van der Waals diameter. 15,16 In addition, hydrogen bonds between backbone carbonyl oxygen CO and amide hydrogen NH groups are represented by virtual moieties located between -carbons, 17 and the chirality is main- tained by dihedral angle potentials. There are several possible explanations for the discrep- ancy between the folding mechanism predicted by all-atom models based on empirical force fields 1–5 and by reduced models. 14,15 One possibility is that folding may not be the reverse of unfolding at high temperature. 18 –20 However, the qualitative agreement between the unfolding results 1 and equilibrium free-energy surface analysis 2,3,5 of all- atom models indicates that the folding mechanism in- ferred from unfolding simulations is likely reliable. An- other possible reason is the inaccuracy of the hydrophobic Grant sponsor: HHMI; Grant sponsor: Center for Computational Research; Grant sponsor: Keck Center for Computational Biology. *Correspondence to: Yaoqi Zhou, HHMI Center for Single Molecule Biophysics, Department of Physiology and Biophysics, State Univer- sity of New York at Buffalo, 124 Sherman Hall, Buffalo, NY 14214. E-mail: yqzhou@buffalo.edu. Received 27 July 2001; Accepted 30 October 2001 Published online XX Month XXXX in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.10065 PROTEINS: Structure, Function, and Genetics 47:154 –162 (2002) © 2002 WILEY-LISS, INC.