FREE-ENERGY PERTURBATIONS IN RIBONUCLEASE-T1 SUBSTRATE-BINDING - A STUDY OF THE INFLUENCE OF SIMULATION LENGTH, INTERNAL DEGREES OF FREEDOMAND STRUCTURE IN FREE-ENERGY PERTURBATIONS

We have studied the reliability of free energy perturbation calculatio ns with respect to simulation protocol and simulation length in a real biological system, the binding of two different ligands to wildtype R ibonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln (RNT1-Gln46). The binding of the natural substrate 3'GMP has been compared the binding of a fluorescent probe, 2-aminopurine 3'mono phos phate (2AP3'MP). These simulations predict that the mutant binds 2AP3' MP better than 3'GMP. Four complete free energy perturbations were pe rformed that form a closed loop of four free energy differences, which should sum up to zero. This could be used as a tool for searching for systematic errors that are not detected by standard forward - backwar d perturbations. The perturbation between 2AP3'MP and 3'GMP is quite s traightforward and similar to what has been done by other groups. The perturbation between Glu46 and Gln46 is much more complex, involving a s many as twelve atoms and a change of charge. This perturbation needs much longer simulation time, 500-600 ps, than used in free energy per turbations before. The increased simulation time is needed both to rea ch an equilibrium and to include several phases of fluctuations of the observed parameters in the production run. The extremely long simulat ion time is not such a severe problem as much of the work might be don e on several different machines in parallel and cheap workstations are excellent for these calculations. Problems may also occur with values of the coupling parameter lambda close to 0 or 1, due to the high mob ility of atoms as well as insertion/deletion in a previously unoccupie d space involved in the perturbation.