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Here, we compare the distributions of main chain ([PHI], [PSI]) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of GlyGly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (ii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the ([PHI], [PSI]) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation, and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the 'intrinsic propensities' of amino acids for particular ([PHI], [PSI]) conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the 'random coil' state. coil library | Dynameomics | molecular dynamics | protein folding | host-guest

The presence of a solvent-exposed alanine residue stabilizes a helix by 0.4-2 kcal * [mol.sup.-1] relative to glycine. Various factors have been suggested to account for the differences in helical propensity, from the higher conformational freedom of glycine sequences in the unfolded state to hydrophobic and van der Waals' stabilization of the alanine side chain in the helical state. We have performed all-atom molecular dynamics simulations with explicit solvent and exhaustive sampling of model peptides to address the backbone conformational entropy difference between Ala and Gly in the denatured state. The mutation of Ala to Gly leads to an increase in conformational entropy equivalent to [approximately equal to] 0.4 kcal 8 [mol.sup.-1] in a fully flexible denatured, that is, unfolded, state. But, this energy is closely counterbalanced by the (measured) difference in free energy of transfer of the glycine and alanine side chains from the vapor phase to water so that the unfolded alanineand glycine--containing peptides are approximately isoenergetic. The helix-stabilizing propensity of Ala relative to Gly thus mainly results from more favorable interactions of Ala in the folded helical structure. The small difference in energetics in the denatured states means that the [PHI]-values derived from Ala [right arrow] Gly scanning of helices are a very good measure of the extent of formation of structure in proteins with little residual structure in the denatured state. folding | pathway | protein | stability | transition state

Several neurodegenerative diseases are linked to expanded repeats of glutamine residues, which lead to the formation of amyloid fibrils and neuronal death. The length of the repeats correlates with the onset of Huntington's disease, such that healthy individuals have 38 repeats exhibit symptoms. Because it is difficult to obtain atomic-resolution structural information for poly(L-glutamine) (polyQ) in aqueous solution experimentally, we performed molecular dynamics simulations to investigate the conformational behavior of this homopolymer. In simulations of 20-, 40-, and 80-mer polyQ, we observed the formation of the '[alpha]-extended chain' conformation, which is characterized by alternating residues in the [[alpha].sub.L] and [[alpha].sub.R] conformations to yield a sheet. The structural transition from disordered random-coil conformations to the [alpha]-extended chain conformation exhibits modest length and temperature dependence, in agreement with the experimental observation that aggregation depends on length and temperature. We propose that fibril formation in polyQ may occur through an [alpha]-sheet structure, which was proposed by Pauling and Corey [Pauling, L. & Corey, R. B. (1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an a-extended chain conformation to inhibit fibril formation. [alpha]-sheet | amyloidosis | misfolding disease | molecular dynamics | poly(L-glutamine)

Molecular dynamics (MD) simulations are the classic single-molecule 'experiments,' providing atomic-resolution structural and dynamic information. However, the single-molecule nature of the technique has also been its shortcoming, with frequent criticisms of sampling inadequacies and questions regarding the ensemble behavior of large numbers of molecules. Given the increase in computer power, we now address this issue by performing a large number of simulations and comparing individual and ensemble properties. One hundred independent MD simulations of the protein chymotrypsin inhibitor 2 were carried out for 20 ns each at 498 K in water to more fully describe the potentially diverse routes of protein unfolding and investigate how representative a single trajectory can be. Rapid unfolding was observed in all Cases with the trajectories distributed about an average 'ensemble' path in which secondary and tertiary structure was lost concomitantly, with tertiary structure loss occurring slightly faster. Individual trajectories did, however, sample conformations far from the average path with very heterogeneous time-dependent properties. Nevertheless, all of the simulations but one followed the average ensemble pathway, such that a small number of simulations (5-10) are sufficient to capture the average properties of these states and the unfolding pathway. energy landscape | folding pathway | molecular dynamics | chymotrypsin inhibitor 2 | unfolding simulations

Transthyretin, [[beta].sub.2]-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of [alpha]-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) Proc. Natl. Acad. Sci. USA 37, 251-256]. In all [beta]-sheet proteins, transthyretin and [[beta].sub.2]-microglobulin, [alpha]-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, [alpha]-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of [alpha]-pleated sheet structure may be a common conformational transition in amyloidosis.

We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental [PHI] values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable [alpha]-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation--condensation. None of the published atomistic simulations are fully consistent with the experimental picture although many capture important features. There is a continuing need for combining simulation with experiment to describe folding pathways, and of continued testing to improve predictive methods. [PHI] value | nucleation-condensation | framework | temperature-jump | kinetics

It is controversial whether fast-folding proteins can form productive on-pathway intermediates that are more stable than the denatured state because noncovalent intermediates are usually evanescent. Here, we apply the classical criteria for the existence of intermediates: namely, the intermediates form and react rapidly enough to be on pathway and they can be isolated and characterized. The folding of the 71-residue, mainly [alpha]-helical FF domain from human HYPA/FBP11 fulfills these classical criteria, as was found for Im7. The FF domain folds in two phases, one on the us and the other on the ms time scale. An engineered mutant folds only to a partly folded state, with some 20-40% of the native helical content, The kinetic properties of the mutant are identical to those found for the fast phase of the wild-type protein, and it is likely that the mutant folds just to the intermediate state. A full kinetic analysis of the folding of wild-type protein, using the amplitudes of its native and denatured states and the observed values for the mutant, rules out an off-pathway scheme but fits an on-pathway scheme, with a low energy intermediate that is modeled by the mutant. The experimental proof benchmarks a molecular dynamics method that identifies an obligatory intermediate observed in multiple simulations. The q space defining this intermediate is visited several times in the simulations, leading to high populations consistent with the presence of a low energy intermediate. FF domain | helix bundle | molecular dynamics | folding intermediate

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.

The ability to diagnose and treat prion diseases is limited by our current understanding of the conversion process of the protein from healthy to harmful isoform. Whereas the monomeric, benign species is well characterized, the misfolded conformations responsible for infectivity and neurodegeneration remain elusive. There is mounting evidence that fibrillization intermediates, or protofibrils, but not mature fibrils or plaques, are the pathogenic species in amyloid diseases. Here, we use molecular dynamics to simulate the conversion of the prion protein. Molecular dynamics simulation produces a scrapie prion protein-like conformation enriched in [beta]-structure that is in good agreement with available experimental data. The converted conformation was then used to model a protofibril by means of the docking of hydrophobic patches of the template structure to form hydrogen-bonded sheets spanning adjacent subunits. The resulting protofibril model provides a non-branching aggregate with a [3.sub.1] axis of symmetry that is in good agreement with a wide variety of experimental data; importantly, it was derived from realistic simulation of the conversion process.

Here, we describe the folding/unfolding kinetics of [[alpha].sub.3]D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 [+ or -] 1.2 [micro]s (at [approximately equal to] 50[degrees]C), indicating that a protein can fold on the 1- to 5-[micro]s time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably [greater than or equal to] 1 [([micro]s).sup.-1] for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. [[alpha].sub.3]D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.

We compare the folding of representative members of a protein superfamily by experiment and simulation to investigate common features in folding mechanisms. The homeodomain superfamily of three-helical, single-domain proteins exhibits a spectrum of folding processes that spans the complete transition from concurrent secondary and tertiary structure formation (nucleation-condensation mechanism) to sequential secondary and tertiary formation (framework mechanism). The unifying factor in their mechanisms is that the transition state for (un)folding is expanded and very native-like, with the proportion and degree of formation of secondary and tertiary interactions varying. There is a transition, or slide, from the framework to nucleation-condensation mechanism with decreasing stability of the secondary structure. Thus, framework and nucleation-condensation are different manifestations of an underlying common mechanism. two-state | three-state | framework | nucleation | homeodomain

Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, [beta]-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal/mol (1 cal = 4.18 J). Preliminary [PHI]-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, [PHI]-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions.

Experimental data from protein engineering studies and NMR spectroscopy have been used by theoreticians to develop algorithms for helix propensity and to benchmark computer simulations of folding pathways and energy landscapes. Molecular dynamic simulations of the unfolding of chymotrypsin inhibitor 2 (CI2) have provided detailed structural models of the transition state ensemble for unfolding/folding of the protein. We now have used the simulated transition state structures to design faster folding mutants of CI2. The models pinpoint a number of unfavorable local interactions at the carboxyl terminus of the single [Alpha]-helix and in the protease-binding loop region of CI2. By removing these interactions or replacing them with stabilizing ones, we have increased the rate of folding of the protein up to 40-fold ([Tau] = 0.4 ms). This correspondence, and other examples of agreement between experiment and theory in general, [Phi]-values and molecular dynamics simulations, in particular, suggest that significant progress has been made toward describing complete folding pathways at atomic resolution by combining experiment and simulation.

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of [10.sup.4] structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix [Alpha]1 (residues 13-17) and in helix [Alpha]2 as well as a turn and nonnative hydrophobic clustering between [Beta]3 and [Beta]4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in [Alpha]1 and the C-terminal portion of the [Beta]-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from [Phi] value analysis and NMR.

Molecular dynamics simulations of the protein unfolding transition state of chymotrypsin inhibitor 2 show that the secondary structure is partially stable while the native tertiary contacts of the hydrophobic core are completely unstable. The ratio of the number of contacts in the residues in the native state to the number in transition state defines the degree of structural modifications in the protein. The transition state for the unfolding process from the simulations agrees with that from protein engineering techniques.

A study was done on the molten globule state using molecular dynamics simulations of protein unfolding and the resulting partially unfolded structures created from bovine pancreatic trypsin inhibitor. The partially denatured products were compact but expanded relative to the native state. The structures were mobile with the overall secondary structure contents resemblingthe native protein. There was local unfolding with the largest changes occurring in the loop regions.

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/ unfolding pathway of CI2 at atomic resolution. CI2 | nuclear magnetic resonance | molecular dynamics simulations | conformational transitions | nucleation-condensation

Under certain conditions, the prion protein (PrP) undergoes a conformational change from the normal cellular isoform, [PrP.sup.C], to [PrP.sup.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 and/or 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.sup.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 [Beta]-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.

The Engrailed Homeodomain protein has the highest refolding and unfolding rate constants directly observed to date. Temperature jump relaxation measurements gave a refolding rate constant of 37,500 [s.sup.-1] in water at 25 [degrees] C, rising to 51,000 [s.sup.-1] around 42 [degrees] C. The unfolding rate constant was 1,100 [s.sup.-1] in water at 25 [degrees] C and 205,000 [s.sup.-1] at 63 [degrees] C. The unfolding half-life is extrapolated to be [approximately equals] 7.5 ns at 100 [degrees] C, which allows real-time molecular dynamics unfolding simulations to be tested on this system at a realistic temperature. Preliminary simulations did indeed conform to unfolding on this time scale. Further, similar transition states were observed in simulations at 100 [degrees] C and 225 [degrees] C, suggesting that high-temperature simulations provide results applicable to lower temperatures. molecular dynamics | fast unfolding | helix docking | residual structure

We present multiple native and denaturation simulations of the B and E domains of the three-helix bundle protein A, totaling 60 ns. The C-terminal helix (H3) consistently denatures later than either of the other two helices and contains residual helical structure in the denatured state. These results are consistent with experiments suggesting that the isolated H3 fragment is more stable than H1 and H2 and that H3 forms early in folding. Interestingly, the denatured state of the B domain is much more compact than that of the E domain. This sequence-dependent effect on the dimensions of the denatured state and the lack of correlation with structure suggest that the radius of gyration can be a misleading reaction coordinate for unfolding/folding. Various unfolding and refolding events are observed in the denaturation simulations. In some cases, the transitions are facilitated through interactions with other portions of the protein--contact-assisted helix formation. In the native simulations, the E domain is very stable: after 6 ns, the [C.sup.[Alpha]] root-mean-square deviation from the starting structure is less than 1.4 [Angstrom]. In contrast, the native state of the B domain deviates more and its inter-helical angles fluctuate. In apparent contrast, we note that the B domain is thermodynamically more stable than the E domain. The simulations suggest that the increased stability of the B domain may be due to heightened mobility, and therefore entropy, in the native state and decreased mobility/entropy in the more compact denatured state.