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SynopsisA simple point-charge potential, developed earlier for the calculation of intermolecular forces in molecular-dynamics simulations of liquid water, has been extended to include interactions between water molecules and polar groups of proteins. A complete potential for use in the simulation of protein dynamics in water is reported.

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We present the first complete treatment for calculating theoretical estimates of free energies of formation of macromolecule-ligand complexes with molecular dynamics simulations, as the free energy for transforming the ligand into a non-interacting state by gradually diminishing the forces between macromolecule (plus solvent) and ligand. The calculations become possible due to the introduction of a specially designed potential ("molecular tweezers") which restrains the spatial position and orientation of the ligand molecule and is gradually applied as the transformation proceeds from complexed to non-interacting components. The binding of benzene to a mutant T4 lysozyme (Morton et al. Biochemistry 1995, 34, 8564-8575) has been used as a test case. The simulations reproduce the value of the free energy of binding (-5.19 kcal/mol if the standard state of benzene is a 1 M aqueous solution) within the sum of experimental and statistical error. Another series of such simulations with rigid protein models provides an estimate of the dependence of the free energy of binding on the protein conformation. The free energy of binding is found to decrease in the series: energy-minimized ligand-free protein (-3.5 kcal/mol), energy-minimized ligand-containing protein (-6.3 kcal/mol), and crystal structure (-8.5 kcal/mol). The free energy of binding to a series of snapshots from a protein-ligand dynamics trajectory varies between -7 and -9 kcal/mol. The "cratic" free energy contribution, which corresponds to the loss of translational and rotational freedom of the ligand molecule, was estimated at 7 kcal/mol. It has proved possible to decompose this into translational and rotational components and, from these free energies, estimate the remaining freedom of the benzene in the binding pocket, at 0.6 Å for positional range and 10-15°for angular range, in excellent agreement with the motion observed in a dynamics trajectory.

The vertebrate immune system has evolved to protect against infections that threaten survival before reproduction. Clinically manifest tumours mostly arise after the reproductive years and somatic mutations allow even otherwise antigenic tumours to evade the attention of the immune system. Moreover, the lack of immunological co-stimulatory molecules on solid tumours could result in T-cell tolerance; that is, the failure of T cells to respond. However, this may not generally apply. Here we report several important findings regarding the immune response to tumours, on the basis of studies of several tumour types. First, tumour-specific induction of protective cytotoxic T cells (CTLs) depends on sufficient tumour cells reaching secondary lymphatic organs early and for a long enough duration. Second, diffusely invading systemic tumours delete CTLs. Third, tumours that stay strictly outside secondary lymphatic organs, or that are within these organs but separated from T cells by barriers, are ignored by T cells but do not delete them. Fourth, co-stimulatory molecules on tumour cells do not influence CTL priming but enhance primed CTL responses in peripheral solid tumours. Last, cross priming of CTLs by tumour antigens, mediated by major histocompatibility complex (MHC) class I molecules of antigen-presenting host cells, is inefficient and not protective. These rules of T-cell induction and maintenance not only change previous views but also rationales for anti-tumour immunotherapy.