SIMULATION OF PROTEIN DYNAMICS AND UNFOLDING IN SOLUTION

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Methods for computer simulation of proteins in solution developed and tested during the first funding period will be applied to systems under active experimental investigation. Our calculation protocol uses a periodic box, includes solvent molecules explicitly, allows full bond length and angle flexibility, truncates long-range interactions smoothly and conserves total energy. It gives well-behaved trajectories that can be computed efficiently. Our newly gained ability to run much longer, more realistic simulations, will help us form strong interactions with experimental collaborators at Standard, NIH, MIT and Los Alamos. Specific aims are: 1. Can we simulate urea and trifluoroethanol? We will molecular dynamics to simulate these denaturing and renaturing solutions at different concentrations and temperatures and calibrate them against experimental data to obtain realistic solutions for use in unfolding and refolding simulations. 2. How do alpha-helix and Beta-hairpin structures unfold? How long does helix unfolding take? What is the nucleation center for helix and hairpin formation? How does Beta-hairpin unfolding, with its long-rang hydrogen bonds, differ from alpha-helix unfolding? These questions will be answered in unfolding simulations lasting 1 to 10 nanoseconds (ns) to be done at a range of temperatures. By reducing temperature slowly, we will also attempt to refold partially denatured alpha-helices and Beta- hairpins. 3. What are the earliest events in protein unfolding? We have carefully chosen a set of five proteins that are (a) small, (b) built according to different architectural principles, and (c) under experimental study. Short simulations (<100ps) will be run at a range of temperatures and densities. Water interactions, packing contacts and packing volume changes will be analyzed to reveal how structure is disrupted. The effects of concentrated urea and TFE will also be investigated. 4. How do unfolding pathways depend on protein architecture? Running prolonged (1-5 ns) simulations at elevated temperatures should caused each of the small proteins studied here to unfold. The nature of the intermediates in unfolding and the way they are formed will be analyzed and compared. 5. How mobile are intermediate states? Dynamics of intermediate states generated by unfolding these five proteins will be run at room temperature. Distances between cysteine, hydrophobic residues and charge pairs will be monitored. 6. Can we restore a native fold to a part of alpha-lactalbumin? We will use our packing methods and simulations to predict stabilizing mutations for a part known to be a molten globule at low temperature.