Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by phi values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native alpha-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion-collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

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Statistical thermodynamic theory has recently been developed to account for the stabilities of globular proteins. Here we extend that work to predict the effects of solvents on protein stability. Folding is assumed to be driven by solvophobic interactions and opposed by conformational entropy. The solvent dependence of the solvophobic interactions is taken from transfer experiments of Nozaki and Tanford on amino acids into aqueous solutions of urea or guanidine hydrochloride (GuHCl). On the basis of the assumption of two pathways involving collapse and formation of a core, the theory predicts that increasing denaturant should lead to a two-state denaturation transition (i.e., there is a stable state along each path separated by a free energy barrier). The denaturation midpoint is predicted to occur at higher concentrations of urea than of GuHCl. At neutral pH, the radius of the solvent-denatured state should be much smaller than for a random-flight chain and increase with either denaturant concentration or number of polar residues in the chain. A question of interest is whether free energies of folding should depend linearly on denaturant, as is often assumed. The free energy is predicted to be linear for urea but to have some small curvature for GuHCl. Predicted slopes and exposed areas of the unfolded states are found to be in generally good agreement with experiments. We also discuss stabilizing solvents and compare thermal with solvent denaturation.

Under certain conditions, the prion protein (PrP) undergoes a conformational change from the normal cellular isoform, PrP C , to PrP Sc , an infectious isoform capable of causing neurodegenerative diseases in many mammals. Conversion can be triggered by low pH, and in vivo this appears to take place in an endocytic pathway and͞or caveolae-like domains. It has thus far been impossible to characterize the conformational change at high resolution by experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed 10-ns molecular dynamics simulations of PrP C in water at neutral and low pH. The core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet-like structure increases both by lengthening of the native ␤-sheet and by addition of a portion of the N terminus to widen the sheet by another two strands. The side chain of Met-129, a polymorphic codon in humans associated with variant Creutzfeldt-Jakob disease, pulls the N terminus into the sheet. Neutralization of Asp-178 at low pH removes interactions that inhibit conversion, which is consistent with the Asp-178 -Asn mutation causing human prion diseases. P rP C is a glycosylated, glycosylphosphatidylinositol-anchored component of the extracellular surface of neurons that appears to play a role in signal transduction (1). PrP Sc , the misfolded isoform, is a ␤-sheet-rich, protease-resistant protein that causes fatal neurodegenerative diseases of the central nervous system in humans and other mammals (2, 3). Clinically, these diseases can exhibit sporadic, inherited, or infectious presentations. Neuropathologically, spongiform degeneration with astrocytic gliosis and extracellular deposits rich in the prion protein (PrP) are observed (4). Inherited disease maps exclusively to mutations in the PrP. Infectious disease is transmitted by PrP Sc , which is chemically indistinguishable from PrP C (5); however, their secondary, tertiary, and quaternary structures differ (6-10). PrP C is monomeric, whereas PrP Sc adopts a multimeric arrangement. Fourier transform infrared and CD spectroscopy studies indicate that PrP C is highly helical (42%), with little ␤-sheet structure (3%) (8). In contrast, PrP Sc contains a large amount of ␤-structure (43%) and less helical structure (30%). These results and others suggest that a conversion of ␣-helices to ␤-sheets is an essential feature in the formation of PrP Sc from PrP C .Recombinant forms of human and murine PrP C undergo a pH-dependent conformational change in the region of pH 4.4-6, with a loss of helix and gain of ␤-structure (11, 12). In vivo, conversion of PrP C 3 PrP Sc is a posttranslational process that appears to occur in an endocytic pathway (7,13,14). Caveolaelike domains have also been implicated in the conversion of wild-type protein (15, 16), and they appear to be acidic (17). Lower pH accelerates conversion in a cell-free conversion assay (10). Thus low pH may play a role in facilitating the conformational cha...