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 residues and individuals with >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(L) and alpha(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. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an alpha-extended chain conformation to inhibit fibril formation.

Transthyretin, beta(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(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.

The homotetramer of transthyretin (TTR) dissociates into a monomeric amyloidogenic intermediate that self-assembles into amyloid fibrils at low pH. We have performed molecular dynamics simulations of monomeric TTR at neutral and low pH at physiological (310 K) and very elevated temperature (498 K). In the low-pH simulations at both temperatures, one of the two β-sheets (strands CBEF) becomes disrupted, and α-sheet structure forms in the other sheet (strands DAGH). α-sheet is formed by alternating αL and αR residues, and it was first proposed by Pauling and Corey. Overall, the simulations are in agreement with the available experimental observations, including solid-state NMR results for a TTR-peptide amyloid. In addition, they provide a unique explanation for the results of hydrogen exchange experiments of the amyloidogenic intermediate—results that are difficult to explain with β-structure. We propose that α-sheet may represent a key pathological conformation during amyloidogenesis.

Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion–collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 μs of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD.

We examined the hydration of amides of alpha(3)D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy ( approximately 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I' band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I' bands of the buried (13)C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3(10)/alpha/pi-helical conformation.

The conformational equilibrium between 3(10)- and alpha-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 3(10)-helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous alphaN(i,i + 2) and alphaN(i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 3(10)- and alpha-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 A and a 10 A force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the (3)J(HNalpha) coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i --> i + 3 hydrogen bonds (3(10)-helix); however, there was a large population of bifurcated i --> i + 3 and i --> i + 4 alpha-helical hydrogen bonds. In addition, both simulations contained considerable populations of pi-helix (i --> i + 5 hydrogen bonds). Individual turns formed over residues 1-9, which we predict contribute to the intensities of the experimentally observed alphaN(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages.

Here, we describe the folding/unfolding kinetics of α 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 ± 1.2 μs (at ≈50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥1 (μs) –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. α 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.

To investigate whether swapping native turns of a globular protein with an elastin-based turn sequence (VPGVG) can increase its thermostability, we have performed molecular dynamics simulations of wild-type chymotrypsin inhibitor 2 (CI2) and variants containing elastin-based turns at 10 degrees C and 40 degrees C. Wild-type CI2 is more stable at 10 degrees C, while both of the variant forms are more stable at 40 degrees C. Detailed analyses indicate that the elastin-based turns do indeed contribute to the inverse temperature behavior of the modified proteins. Therefore, swapping a wild-type turn sequence with an elastin-based turn provides a novel way to both improve stability of target proteins at body temperature and to possibly introduce a temperature-sensitive switch.

As computer power increases, so too does the range of interesting biomolecular phenomena and properties that can be simulated. It is now possible to simulate complicated protein conformational changes at ambient or physiological temperatures. In this regard, we are attempting to map the conformational transitions of the normal, cellular prion protein (PrP(C)) to its infectious scrapie isoform (PrP(Sc)), which causes neurodegenerative diseases in many mammals. These two forms have identical sequences and are conformational isomers, with heightened formation of beta-sheet structure in the scrapie form. Conversion can be triggered by lowering the pH, but thus far it has been impossible to characterize the conformational change at high resolution using experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed molecular-dynamics simulations of hamster, human and bovine forms of the prion protein in water at neutral and low pH. In all cases 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 2-3 strands.

Previous molecular dynamics (MD) simulations of the thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided atomic-resolution models of the transition state ensemble that is well supported by experimental studies. Here, we use simulations to further investigate the energy landscape around the transition state region. Nine structures within ∼35 ps and 3 Å Cα RMSD of the transition state ensemble identified in a previous 498 K thermal denaturation simulation were quenched under the quasi-native conditions of 335 K and neutral pH. All of the structures underwent hydrophobically driven collapse in response to the drop in temperature. Structures less denatured than the transition state became structurally more native-like, while structures that were more denatured than the transition state tended to show additional loss of native structure. The structures in the immediate region of the transition state fluctuated between becoming more and less native-like. All of the starting structures had the same native-like topology and were quite similar (within 3.5 Å Cα RMSD). That the structures all shared native-like topology, yet diverged into either more or less native-like structures depending on which side of the transition state they occupied on the unfolding trajectory, indicates that topology alone does not dictate protein folding. Instead, our results suggest that a detailed interplay of packing interactions and interactions with water determine whether a partially denatured protein will become more native-like under refolding conditions.

The structure and dynamics of the water hydrating peptides and proteins are examined here at atomic resolution via molecular dynamics simulations. Detailed solvation density and residence time data for all 20 l-amino acids in an end-capped AXA tripeptide motif are presented. In addition, the solvation of the protein chymotrypsin inhibitor 2 is investigated as a point of comparison. Residues on the surface of proteins are not isolated; they interact both locally and non-locally in sequence space, and comparison of the solvation properties of each amino acid in both the peptide and protein allow us to distinguish inherent solvation properties from context-dependent perturbations due to neighboring residues. This work moves beyond traditional radial distribution functions and presents graphical representations of preferential solvation and orientation of water by side chains and the main chain. The combination of 0.3 μs of simulation data improves the statistical sampling over previous studies and reveals the significance of bridging water molecules that stabilize and mediate side chain–side chain, side chain–main chain and main chain–main chain interactions at the solvation interface.

Molecular dynamics simulations with explicit waters have been employed to investigate the dominant source of elastin's elasticity. An elastin-like peptide, (VPGVG)(18), was pulled and released in molecular dynamics simulations, at 10 and 42 degrees C, lasting several nanoseconds, which is consistent with the experimentally determined dielectric and NMR relaxation time scales. At elastin's physiological temperature and degree of extension, the simulations indicate that the orientational entropy of waters hydrating hydrophobic groups decreases during pulling of the molecule, but it increases upon release. In contrast, the main-chain fluctuations and other measures of mobility suggest that elastin's backbone is more dynamic in the extended than released state. These results and the agreement between the simulations with various experimental observations suggest that hydrophobic hydration is an important source of the entropy-based elasticity of elastin. Moreover, elastin tends to reorder itself to form a hydrophobic globule when it was held in its extended state, indicating that the hydrophobic effect also contributes in the holding process. On the whole, our simulations support the hydrophobic mechanism of elasticity and provide a framework for description of the molecular basis of this phenomenon.

Elastin undergoes an "inverse temperature transition" such that it becomes more ordered as the temperature increases. To investigate the molecular basis for this behavior, molecular dynamics simulations were conducted above and below the transition temperature. Simulations of a 90-residue elastin peptide, (VPGVG)(18), with explicit water molecules were performed at seven different temperatures between 7 and 42 degrees C, for a total of 80 ns. Beginning from an idealized beta-spiral structure, hydrophobic collapse was observed over a narrow temperature range in the simulations. Moreover, simulations above and below elastin's transition temperature indicate that elastin has more turns and distorted beta-structure at higher temperatures. Water was critical to the inverse temperature transition and elastin-associated water molecules can be divided into three categories: those closely associated with beta II turns; those that form hydrogen bonds with the main-chain groups; and those hydrating the hydrophobic side-chains. Water-swollen, monomeric elastin above the transition temperature is best described as a compact amorphous structure with distorted beta-strands, fluctuating turns, buried hydrophobic residues, and main-chain polar atoms that participate in hydrogen bonds with water. Below the transition temperature, elastin is expanded with approximately 40 % local beta-spiral structure. Overall the simulations are in agreement with experiment and therefore appear to provide an atomic-level description of the conformational properties of elastin monomers and the basis for their elastomeric properties.

Publisher Summary Prions are transmissible pathogens that cause fatal neurodegenerative diseases of the central nervous system in humans and animals. In humans, prion diseases have been subdivided into four classes and are referred to as kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI). These diseases can be sporadic, inherited, or infectious (or iatrogenic) disorders. They differ from other infectious diseases, in that the pathogen is a proteinaceous particle, termed a “prion,” and the essential component of prions is the scrapie prion protein (PrP Sc ).

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 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 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 β-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.

Background: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle.Results: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha 1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition stale, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta 2 and beta 4 remain associated.Conclusions: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta 2-beta 4 residual structure is surprising, because beta 4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.

Nine nonnative conformations of ubiquitin, generated during two different thermal denaturation trajectories, were simulated under nearly native conditions (62 °C). The simulations included all protein and solvent atoms explicitly, and simulation times ranged from 1‐2.4 ns. The starting structures had α‐carbon root‐mean‐square deviations (RMSDs) from the crystal structure of 4‐12 Å and radii of gyration as high as 1.3 times that of the native state. In all but one case, the protein collapsed when the temperature was lowered and sampled conformations as compact as those reached in a control simulation beginning from the crystal structure. In contrast, the protein did not collapse when simulated in a 60% methanol: water mixture. The behavior of the protein depended on the starting structure: during simulation of the most native‐like starting structures (≤5 Å RMSD to the crystal structure) the RMSD decreased, the number of native hydrogen bonds increased, and the secondary and tertiary structure increased. Intermediate starting structures (5‐10 Å RMSD) collapsed to the radius of gyration of the control simulation, hydrophobic residues were preferentially buried, and the protein acquired some native contacts. However, the protein did not refold. The least native starting structures (10‐12 Å RMSD) did not collapse as completely as the more native‐like structures; instead, they experienced large fluctuations in radius of gyration and went through cycles of expansion and collapse, with improved burial of hydrophobic residues in successive collapsed states.

Background: Prion diseases are neurodegenerative disorders that appear to be due to a conformational change, involving the conversion of alpha-helices in the normal, cellular isoform of the prion protein (PrPC) to beta-structure in the infectious scrapie form (PrPSc). One form of Gerstmann-Straussler-Scheinker syndrome (GSS), an inherited prion disease, is caused by mutation of Ala117 of PrPC to Val. We therefore set out to evaluate the effects of this mutation on the stability of the PrPC form.Results: We have performed molecular dynamics simulations of a portion of the PrPC sequence (residues 109-122, termed H1) that is proposed to figure prominently in the conversion of PrPC to PrPSc. In particular, beginning with H1 in the alpha-helical state, the conformational consequences of sequence changes at position 117 were investigated for six hydrophobic mutations. Of these, only the Val mutation was helix-destabilizing. Portions of this mutant peptide adopted and retained an extended conformation during a 2 ns simulation of the peptide in water.Conclusions: The conformational transitions and structures observed in the simulation of the mutant peptide with Val at position 117 provide insight into the possible early steps in the conversion of PrPC to PrPSc.

Extensive experimental data are available on the native, partially and fully unfolded states of ubiquitin. Two and three-dimensional NMR experiments of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains disrupted but native-like structure while the other half is unstructured and/or contains non-native structure. In contrast, the interpretation of hydrogen-exchange data have led to the conclusion that this state is native-like. Thus, there are discrepancies between the experimental studies, or interpretations based on the data. We compare the results of molecular dynamics simulations, under varying conditions, with the experimental results. The simulations extend past 0.5 ns and include explicit solvent molecules: either pure water or 60% methanol. To begin with, ubiquitin was thermally denatured in water (at 498 K). Two particular structures, or "aliquots", during the unfolding process were selected for further study (60 and 198 ps). These structures were then simulated separately in water and 60% methanol at a lower and experimentally meaningful temperature (335 K). The conformations generated from the structure extracted later in the simulation contained significant amounts of non-native structure in the presence of methanol while satisfying both the NMR and hydrogen exchange data. In fact, clearly non-native regions of the structure yielded the desired protection from hydrogen exchange. In contrast, an earlier, more native-like, intermediate did not do as well at predicting the hydrogen-exchange behavior and was inconsistent with the NMR data. These data suggest that the results and interpretations using the different experimental techniques can be reconciled by a single state. This finding also brings into question the practice of interpreting protection to hydrogen exchange in terms of native secondary and tertiary structure, especially when one has weak patterns and low protection factors. When the partially unfolded states were placed in pure water, the protein collapsed and began to refold. Therefore, the desired solvent-dependent properties were observed: the partially unfolded conformations with increased exposure of hydrophobic residues remained expanded in methanol but collapsed in water as the non-polar groups minimized their exposure to solvent.

We develop mean-field lattice statistical mechanics theory for the equilibrium between denatured and aggregated states of proteins and other random copolymers of hydrophobic and polar monomers in aqueous solution. We suppose that the aggregated state is a mixture of amorphous polymer plus solvent and that the driving forces are the hydrophobic interaction, which favors aggregation, and conformational and translational entropies, which favor disaggregation. The theory predicts that the phase diagram for thermal aggregation is an asymmetric closed loop, and for denaturants (guanidinium hydrochloride or urea) it is asymmetric with an upper consolute point. The theory predicts that a copolymer in a poor solvent will expand with increasing polymer concentration because of "screening" of the solvent interactions by the other chains; the chain ultimately reaches a theta-like state in the absence of solvent. The screening concentration depends strongly on the copolymer composition. We find two striking features of these copolymer phase diagrams. First, they are extraordinarily sensitive to the copolymer composition; a change of one amino acid can substantially change the aggregation behavior. Second, relative to homopolymers, copolymers should be stable against aggregation at concentrations that are higher by many orders of magnitude.