Calculations on Folding of Segment B1 of Streptococcal Protein G Felix B. Sheinerman and Charles L. Brooks III* Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA We present an investigation of the folding thermodynamics and mechan- ism of segment B1 of streptococcal protein G. Molecular dynamics simu- lations of the fully solvated protein are used to probe thermodynamically signi®cant states at different stages of folding. We performed several unfolding simulations to generate a database of initial conditions. The database is analyzed and clustered. The cluster centers extracted from this database were then used as starting points for umbrella sampling of the folding free energy landscape under folding conditions. The resulting sampling was combined with the weighted histogram analysis method. One and two-dimensional free energy surfaces were constructed along several order parameters and used to analyze the folding process. Our ®ndings indicate that an initial collapse precedes the formation of signi®- cant native structure. Elements of local structure originate in the regions of the protein shown to have higher H/ 2 H exchange protection factors in early stages of folding. A non-native contact, observed experimentally at the N terminus of the a-helix in a peptide excised from the protein, is seen to pre-organize the chain in early stages of folding. Collapse and early structure formation yields a compact globule with a signi®cant number of water molecules present. Desolvation of the protein core is coincident with the ®nal stages of folding from the compact state. # 1998 Academic Press Limited Keywords: protein folding; folding mechanism; potential of mean force; molecular dynamics; protein G *Corresponding author Introduction Since An®nsen elucidated the nature of the pro- tein folding problem (An®nsen, 1973), understand- ing the sequence requirements for the development of a speci®c ®nal folded structure, quests for the ``rules'' that determine this sequence to structure process as well as the physical principles that gov- ern the formation of the native conformation have been underway. The physical side of the protein folding problem, investigation of the forces and mechanisms governing formation of the native conformation, is extremely important for the gener- al understanding of protein function. As protein function is de®ned by protein structure, the ability to predict conformational transitions caused by changes in external conditions is essential to our understanding of protein function. Recent years have witnessed signi®cant advance- ments in our understanding of the physical side of protein folding. These advances have stemmed from both experimental and theoretical progress. Developments on the theory side are based on both advances in analytical treatments of the phenomenon of folding and the elaboration of numerical simulation techniques (Bryngelson & Wolynes, 1987; Dill et al., 1995; Guo & Brooks, 1997; Guo & Thirumalai, 1995; Klimov & Thirumalai, 1996; Leopold et al., 1992; Luthey- Schulten et al., 1995; Sali et al., 1994; Saven & Wolynes, 1996; Shakhnovich, 1997; Shakhnovich & Gutin, 1989; Shoemaker et al., 1997). The theoretical developments have lead to the formulation of a ``new'' view of protein folding (Dill & Chan, 1997; Onuchic et al., 1997). This view focuses on the description of general statistical properties of the energy landscape of a protein rather than the characterization of speci®c intermediates occurring during the folding reaction. The evolution of theoretical concepts has been stipulated by the development of experimental techniques for monitoring fast events and detect- Abbreviations used: GB1, segment B1 of streptococcal protein G; NOE, nuclear Overhauser enhancement; CI2, chymotrypsin inhibitor 2; GuHCl, guanidine-HCl; MD, molecular dynamics; PMF, potential mean force. J. Mol. Biol. (1998) 278, 439±456 0022±2836/98/170439±18 $25.00/0/mb981688 # 1998 Academic Press Limited