New atom-and group-based spherical-cutoff methods have been developed for the treatment of nonbonded interactions in molecular dynamics (MD) simulation. A new atom-based method, force switching, leaves short-range forces unaltered by adding a constant to the potential energy, switching forces smoothly to zero over a specified range. A simple improvement to group-based cutoffs is presented: Switched group-shifting shifts the groupgroup potential energy by a constant before being switched smoothly to zero. Also introduced are generalizations of atom-based force shifting, which adds a constant to the Coulomb force between two charges. These new approaches are compared to existing methods by evaluating the energy of a model hydrogen-bonding system consisting of two N-methyl acetamide molecules and by full MD simulation. Thirty-five 150 ps simulations of carboxymyoglobin (MbCO) hydrated by 350 water molecules indicate that the new methods and atom-based shifting are each able to approximate no-cutoff results when a cutoff at or beyond 12 8, is used. However, atom-based potential-energy switching and truncation unacceptably contaminate groupgroup electrostatic interactions. Group-based potential truncation should not be used in the presence of explicit water or other mobile electrostatic dipoles because energy is not a state function with this method, resulting in severe heating (about 4 K/ps in the simulations of hydrated MbCO). The distancedependent dielectric ( E 0: r) is found to alter the temperature dependence of protein dynamics, suppressing anharmonic motion at high temperatures. Force switching and force shifting are the best atom-based spherical cutoffs, whereas switched group-shifting is the preferred group-based method. To achieve realistic simulations, increasing the cutoff distance from 7.5 to 12 A or beyond is much more important than the differences among the three best cutoff methods.

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To determine whether Fö rster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Fö rster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Fö rster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R 0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Fö rster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donoracceptor distances.Fö rster resonance energy transfer ͉ molecular dynamics ͉ polypeptide ͉ FRET A lmost 40 years ago, Förster resonance energy transfer (FRET) was introduced in classic experiments by Stryer and Haugland (1) as a ''spectroscopic ruler'' to measure distances in macromolecules. Since then it has been used to address a wide range of biological questions (2-5). More recently, renewed interest has come from the realization that FRET can be used for obtaining distance information in experiments on single biomolecules (6, 7), with a considerable body of work on proteins and polypeptides (8 -24). However, it is well known from ensemble experiments that determination of distances from FRET can be complicated by dynamical effects as well as photophysical, photochemical, and instrumental factors (25). Are there additional complications in single-molecule experiments on polypeptides and proteins? To investigate this question, we studied FRET between dyes attached to the N and C termini of polyproline of various lengths. Polyproline, assumed to be a rigid rod, was used as a spacer by Stryer and Haugland to show that the rate of FRET depends on the inverse sixth power of the donor-acceptor distance, as predicted by Förster theory (26).FRET of individual dye-labeled polyproline molecules freely diffusing in solution was investigated by using a confocal f luorescence microscope setup (13). If a molecule diffuses into the volume illuminated by the focused laser beam, the donor dye is excited. Depending on the distance to the acceptor, a certain rate of energy transfer results, which determines the FRET efficiency, calculated from the fraction of photons emitted by the acceptor. To test the accuracy of the singlemolecule results, we also determined FRET efficiencies from ensemble measurements of donor lifetimes in the pres...