Chignolin is an artificial mini-protein composed of 10 residues (GYDPETGTWG) that has been shown to cooperatively fold into a b-hairpin structure in water. We extensively explored the conformational space of chignolin using a 180-ns multicanonical molecular dynamics (MD) simulation and analyzed its folding free-energy landscape. In the MD trajectory, we found structures that satisfy 99% of the experimental restraints and are quite close to the experimentally determined structures with C a root-meansquare-deviations of less than 0.5 Å . These structures formed a large cluster in the conformational space with the largest probability of existence, agreeing well with the experiment.

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Hydrophobic effects in aqueous urea were analyzed by molecular dynamics simulations. The contribution of solvents to the potential of mean force between two methane molecules was calculated by using molecular dynamics simulations and was compared with the solubility data of hydrocarbons in aqueous urea. Both the simulation results and the solubility data indicated that urea stabilizes methane-methane association. The stabilization was due to increasing the solvation free energies of small hydrocarbons such as methane by addition of urea. The solvation free energies of larger hydrocarbons, on the other hand, are decreased by addition of urea. This effect of the solute size on hydrophobic free energies in aqueous urea was also analyzed by using molecular dynamics simulations by means of division of the solvation process into two parts: the cavity formation and the introduction of the solute-solvent attractive interactions. In the cavity formation, urea increased hydrophobic free energies, and in the introduction of the solute-solvent attractive interactions, urea decreased hydrophobic free energies. The influence of urea on hydrophobic free energies was determined by the balance of effects of the two parts of the solvation process.

Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, we truncated the human EGFR just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992 -* Phe (DEL1011+F992). The wild-type and mutant receptors were stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. As expected, DEL1011 and DEL1011+F992 were found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still could induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these was significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C yl was dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. On the other hand, tyrosine phosphorylation of Shc, complex formation of ShcGrb2/Ash, and activation of microtubule-associated protein kinase were still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant.

Because functional analysis of Fc receptors (FcRs) relies heavily on mouse models, the identification of another Fcgamma receptor is particularly noteworthy. We demonstrate that FcgammaRIV, identified here as the mouse ortholog of primate FcgammaRIII, required association of the FcR gamma-chain for optimal expression and function on myeloid cells; its signaling potential was also enhanced by a cytoplasmic 'YEEP' motif that was able to recruit the adaptor molecule Crk-L and phosphatidylinositol-3-OH kinase. Unexpectedly, FcgammaRIV 'preferentially' bound immunoglobulin E antibodies of the 'b' allotype (IgE(b)) as well as IgG2a and IgG2b antibodies. Ligation of FcgammaRIV by antigen-IgE(b) immune complexes promoted macrophage-mediated phagocytosis, presentation of antigen to T cells, production of proinflammatory cytokines and the late phase of cutaneous allergic reactions. IgE(b) antibody-mediated modification of macrophage responses may therefore influence mouse asthma models and strain-dependent differences in parasite susceptibility.