All-atom computer simulations of amyloid fibrils disaggregation

Amyloidlike fibrils are found in many fatal diseases, including Alzheimer's disease, type II diabetes mellitus, transmissible spongiform encephalopathies, and prion diseases. These diseases are linked to proteins that have partially unfolded, misfolded, and aggregated into amyloidlike fibrils. The kinetics of amyloidlike fibrils aggregation is still hotly debated and remains an important open question. We have utilized the GNNQQNY crystal structure and high-temperature molecular dynamics simulation in explicit solvent to study the disaggregation mechanism of the GNNQQNY fibrils and to infer its likely aggregation pathways. A hexamer model and a 12-mer model both with two parallel β-sheets separated by a dry side-chain interface were adopted in our computational analysis. A cumulative time of 1 μs was simulated for the hexamer model at five different temperatures (298 K, 348 K, 398 K, 448 K, and 498 K), and a cumulative time of 2.1 μs was simulated for the 12-mer model at four temperatures (298 K, 398 K, 448 K, and 498 K). Our disaggregation landscape and kinetics analyses indicate that tetramers probably act as the transition state in both the hexamer and the 12-mer simulations. In addition, the 12-mer simulations show that the initial aggregation nucleus is with eight peptides. Furthermore, the landscape is rather flat from 8-mers to 12-mers, indicating the absence of major barriers once the initial aggregation nucleus forms. Thus, the likely aggregation pathway is from monomers to the initial nucleus of 8-mers with tetramers as the transition state. Transition state structure analysis shows that the two dominant transition state conformations are tetramers in the 3-1 and 2-2 arrangements. The predominant nucleus conformations are in peptide arrangements maximizing dry side-chain contacts. Landscape and kinetics analyses also indicate that the parallel β-sheets form earlier than the dry side-chain contacts during aggregation. These results provide further insights in understanding the early fibrils aggregation.     

Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.     

A variational model for oligomer-formation process of GNNQQNY peptide from yeast prion protein Sup35

Many human neurodegenerative diseases are associated with the aggregation of insoluble amyloid-like fibrous proteins. However, the processes by which the randomly diffused monomer peptides aggregate into the highly regulated amyloid fibril structures are largely unknown. We proposed a residue-level coarse-grained variational model for the investigation of the aggregation pathway for a small assembly of amyloid proteins, the peptide GNNQQNY from yeast prion protein Sup35. By examining the free energy surface, we identified the residue-level sequential pathways for double parallel and antiparallel β-peptides, which show that the central dry polar zipper structure is the major folding core in both cases. The critical nucleus size is determined to be three peptides for the homogeneous nucleation process, whereas the zig-zag growth pattern appears most favorably for heterogeneous nucleation. Consistent with the dock-and-lock mechanism, the aggregation process of free peptides to the fibril core was found to be highly cooperative. The quantitative validation with the computational simulations and experimental data demonstrated the usefulness of the proposed model in understanding the general mechanism of the amyloid fibril system.     

GNNQQNY—Investigation of Early Steps during Amyloid Formation

Protein aggregation has been implicated in the pathology of several neurodegenerative diseases, and a better understanding of how it proceeds is essential for the development of therapeutic strategies. Recently, the amyloidogenic heptapeptide GNNQQNY has emerged as a molecule of choice for fundamental studies of protein aggregation. A number of experimental and computational studies have examined the structure of the GNNQQNY aggregate. Less work, however, has been aimed at understanding its aggregation pathway. In this study, we present a detailed computational analysis of such a pathway. To that end, transition path sampling Monte Carlo simulations are used to examine the dimerization process. A statistical analysis of the reaction pathways shows that the dimerization reaction proceeds via a zipping mechanism, initiated with the formation of distinct contacts at the third residue (N). Asparagine residues are found to play a key role in the early stages of aggregation. And, contrary to previous belief, it is also shown that the tyrosine terminal group is not required to stabilize the dimer. In fact, an asparagine residue leads to faster aggregation of the peptide.     

Thermodynamic analysis of structural transitions during GNNQQNY aggregation

Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutzfeldt‐Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full‐length amyloid proteins is not necessary for understanding amyloid formation. In this study, we simulate GNNQQNY, the N‐terminal prion‐determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. Utilizing a coarse‐grained model permits equilibration on relevant time scales. Replica‐exchange molecular dynamics is used to gather simulation statistics at multiple temperatures and clear energy traps that would aversely impact results. Investigating the association of 3‐, 6‐, and 12‐chain GNNQQNY systems by calculating thermodynamic quantities and orientational order parameters, we determine the aggregation pathway by studying aggregation states of GNNQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H‐bond formation, leading to the formation of β‐sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak. Proteins 2013; 81:1141–1155. © 2013 Wiley Periodicals, Inc.     

Single-molecule Force Spectroscopy Predicts a Misfolded,Domain-swapped Conformation in human γD-Crystallin Protein

Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the β1 and β2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.     

Reverse engineering an amyloid aggregation pathway with dimensional analysis and scaling

Human islet amyloid polypeptide (hIAPP) is a cytotoxic protein that aggregates into oligomers and fibrils that kill pancreatic β-cells. Here we analyze hIAPP aggregation in vitro, measured via thioflavin-T fluorescence. We use mass-action kinetics and scaling analysis to reconstruct the aggregation pathway, and find that the initiation step requires four hIAPP monomers. After this step, monomers join the nucleus in pairs, until the first stable nucleus (of size approximately 20 monomers) is formed. This nucleus then elongates by successive addition of single monomers. We find that the best-fit of our data is achieved when we include a secondary fibril-dependent nucleation pathway in the reaction scheme. We predict how interventions that change rates of fibril elongation or nucleation rates affect the accumulation of potentially cytotoxic oligomer species. Our results demonstrate the power of scaling analysis in reverse engineering biochemical aggregation pathways.     

Sequence-dependent conformational differences of small RNAs revealed by native gel electrophoresis.

In this study, we use native polyacrylamide gel electrophoresis and one-dimensional NMR spectroscopy to analyze small RNA hairpins containing a UUCG tetraloop. The aggregation state of one RNA 16-mer (5'-CGGCUUCGGUCGACCA-3') in the presence of Mg(2+) was confirmed by laser light scattering. Although it is widely known in the RNA field that some RNAs tend to aggregate, especially when present at high concentrations, the sequence elements responsible for this effect are rarely identified. In this work, we show that Mg(2+)-induced aggregation of the 16-mer RNA hairpin is sensitive to the presence of the 3'-terminal base and a specific 2'-hydroxyl group. Our study highlights the fact that even small changes in a particular RNA sequence can increase its tendency to undergo Mg(2+)-dependent aggregation in an unpredictable manner. Our analysis also shows that native gel electrophoresis is a sensitive probe of RNA conformation with the capability to detect differences apparently caused by subtle base stacking effects at the ends of helices.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Supramolecular self-assembly of d(TGG)4, synergistic effects of K+ and Mg2+.

Spectral evidence indicates that molar concentrations of K+ can induce aggregate formation in d(TGG)4. The 320-nm turbidity monitoring indicates that more than 1 M KCl is needed for the onset of aggregation to occur at 20 degrees C within the time span of 24 h. The kinetic profile is reminiscent of autocatalytic reactions that consist of a lag period followed by accelerative and levelling phases. Progressive shortening of lag periods and more rapid accelerative phases accompany further increases in [K+]. Interestingly, the presence of Mg2+ greatly facilitates the aggregate formation and results in the prominent appearance of an intense psi-type CD. For example, whereas 1 M K+ fails to induce aggregate formation of d(TGG)4 within 24 h, the addition of 1 mM Mg2+ to a 1 M K+ solution is sufficient to induce the onset of aggregation in approximately 12 h. Furthermore, adjustment of the buffer to 16 mM Mg2+/1 M KCl reduces the lag time to less than 10 min and aggregation is nearly complete in 2 h. The requirement of [K+] for aggregation is reduced to 2 mM in the presence of 16 mM Mg2+, a reduction of nearly three orders of magnitude when compared to solutions without Mg2+. The effects of K+ and Mg2+ ions are synergistic, because the presence of 16 mM Mg2+ alone does not induce aggregate formation in this oligomer. Thermal stabilities of the aggregates are strongly dependent on the concentrations of these two ions. Although aggregates formed in the presence of 2 M KCl alone melt around 55 degrees C, those formed with added 16 mM Mg2+ melt at approximately 90 degrees C, with some aggregates remaining unmelted even at 95 degrees C. The slow kinetics of aggregate formation led to the appearance of gross hystereses in the cooling profiles. The interplay of these two ions appears to be specific, because the replacement of K+ by Na+ or the replacement of Mg2+ by other divalent cations does not lead to the observed self-assembly phenomenon, although Sr2+ can substitute for K+. A possible mechanism for the formation of self-assembled structures is suggested.     

Size, orientation and organization of oligomers that nucleate amyloid fibrils: Clues from MD simulations of pre-formed aggregates

All-atom MD simulations of pre-formed aggregates of GNNQQNY with variable size (5 to 16 peptides), orientation (parallel or anti-parallel), organization (single or double sheet, with or without twist), charge status of termini and temperature (300 and 330K) have been performed for 50ns each (68 simulations; total time=3.4μs). Double-layer systems are stable irrespective of whether the peptides within the sheet are oriented parallel or anti-parallel. The lifetime of single sheet systems is determined by the protonation status, nature of association of peptides and the size of the aggregates. For example, single sheet 8-mers are stable with parallel arrangement and neutral termini, or with anti-parallel arrangement and charged termini. This suggests that the residues flanking the amyloidogenic sequence also play an important role in determining the organization of peptides in an aggregate. Twist of the cross-beta sheets is found to be intrinsic to the aggregates. Main chain H-bonds are key determinants of stability and loss of these H-bonds is followed by disorder and/or dissociation of the peptide despite the presence of side chain hydrogen bonds. Aggregates are inherently asymmetric along the fiber axis and dissociation from the C-edge is observed more often. An aggregate can disintegrate into smaller-sized oligomers or the edge peptides can dissociate sequentially. A variety of dissociation and disintegration events are observed pointing to the existence of multiple pathways for association during nucleation. It appears that a heterogeneous mixture of oligomers of different sizes exist prior to the formation of the critical nucleus.     

On the Characterization of Intermediates in the Isodesmic Aggregation Pathway of Hen Lysozyme at Alkaline pH

Protein aggregation leading to formation of amyloid fibrils is a symptom of several diseases like Alzheimer’s, type 2 diabetes and so on. Elucidating the poorly understood mechanism of such phenomena entails the difficult task of characterizing the species involved at each of the multiple steps in the aggregation pathway. It was previously shown by us that spontaneous aggregation of hen-eggwhite lysozyme (HEWL) at room temperature in pH 12.2 is a good model to study aggregation. Here in this paper we investigate the growth kinetics, structure, function and dynamics of multiple intermediate species populating the aggregation pathway of HEWL at pH 12.2. The different intermediates were isolated by varying the HEWL monomer concentration in the 300 nM—0.12 mM range. The intermediates were characterized using techniques like steady-state and nanosecond time-resolved fluorescence, atomic force microscopy and dynamic light scattering. Growth kinetics of non-fibrillar HEWL aggregates were fitted to the von Bertalanffy equation to yield a HEWL concentration independent rate constant (k = (6.6±0.6)×10⁻⁵ s⁻¹). Our results reveal stepwise changes in size, molecular packing and enzymatic activity among growing HEWL aggregates consistent with an isodesmic aggregation model. Formation of disulphide bonds that crosslink the monomers in the aggregate appear as a unique feature of this aggregation. AFM images of multiple amyloid fibrils emanating radially from amorphous aggregates directly confirmed that on-pathway fibril formation was feasible under isodesmic polymerization. The isolated HEWL aggregates are revealed as polycationic protein nanoparticles that are robust at neutral pH with ability to take up non-polar molecules like ANS.     

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K(4)K(20)-S4 was 2-3-fold more potent against protozoa and RBC, yet K(4)K(20)-S4 was more potent by 2 orders of magnitude against bacteria. K(4)-S4 had similar behavior as K(4)K(20)-S4, but K(20)-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K(20)-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K(4)-S4 and K(4)K(20)-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.     

The Lys 280 → Gln mutation mimicking disease‐linked acetylation of Lys 280 in tau extends the structural core of fibrils and modulates their catalytic properties

Amyloid fibrillar aggregates isolated from the brains of patients with neurodegenerative diseases invariably have post‐translational modifications (PTMs). The roles that PTMs play in modulating the structures and polymorphism of amyloid aggregates, and hence their ability to catalyze the conversion of monomeric protein to their fibrillar structure is, however, poorly understood. This is particularly true in the case of tau aggregates, where specific folds of fibrillar tau have been implicated in specific tauopathies. Several PTMs, including acetylation at Lys 280, increase aggregation of tau in the brain, and increase neurodegeneration. In this study, tau‐K18 K280Q, in which the Lys 280 → Gln mutation is used to mimic acetylation at Lys 280, is shown, using HX‐MS measurements, to form fibrils with a structural core that is longer than that of tau‐K18 fibrils. Measurements of critical concentrations show that the binding affinity of monomeric tau‐K18 for its fibrillar counterpart is only marginally more than that of monomeric tau‐K18 K280Q for its fibrillar counterpart. Quantitative analysis of the kinetics of seeded aggregation, using a simple Michaelis–Menten‐like model, in which the monomer first binds and then undergoes conformational conversion to β‐strand, shows that the fibrils of tau‐K18 K280Q convert monomeric protein more slowly than do fibrils of tau‐K18. In contrast, monomeric tau‐K18 K280Q is converted faster to fibrils than is monomeric tau‐K18. Thus, the effect of Lys 280 acetylation on tau aggregate propagation in brain cells is expected to depend on the amount of acetylated tau present, and on whether the propagating seed is acetylated at Lys 280 or not.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Polyglutamine expansion mutation yields a pathological epitope linked to nucleation of protein aggregate: determinant of Huntington's disease onset

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells.     

Folding nucleus structure persists in thermally‐aggregated FGF‐1

An efficient protein‐folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally‐induced aggregate of fibroblast growth factor‐1 (FGF‐1), a small globular protein, by solid‐state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a structured region that corresponds to the region of the folding nucleus. NMR data on aggregated FGF‐1 also indicate the presence of unstructured regions that exhibit hydration‐dependent dynamics and suggest that unstructured regions of aggregated FGF‐1 lie outside the folding nucleus. Since it is known that regions outside the folding nucleus fold late in the folding pathway, we postulate that these regions unfold early in the unfolding pathway and that the partially folded state is more prone to intermolecular aggregation. This interpretation is further supported by comparison with a designed protein that shares the same FGF‐1 folding nucleus sequence, but has different 1° structure outside the folding nucleus, and does not thermally aggregate. The results suggest that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria – the latter of which could principally focus upon the physicochemical properties of 1° structure outside the folding nucleus.     

Stability of single sheet GNNQQNY aggregates analyzed by replica exchange molecular dynamics: antiparallel versus parallel association

Protein and peptide aggregation into amyloid plaques is associated with a large variety of neurodegenerative diseases. The definition of the molecular bases of these pathologies is hampered by the transient nature of pre-fibrillar small-oligomers that are considered the toxic species. The ability of the peptide GNNQQNY to form amyloid-like structures makes it a good model to investigate the complex processes involved into amyloid fiber formation. By employing full atomistic replica exchange molecular dynamics simulations, we constructed the free energy surface of small assemblies of GNNQQNY to gain novel insights into the fiber formation process. The calculations suggest that the peptide exhibits a remarkable tendency to form both parallel and antiparallel β-sheets. The data show that GNNQQNY preference for parallel or antiparallel β-sheets is governed by a subtle balance of factors including assemblies’ size, sidechain-sidechain interactions and pH. The samplings analysis provides a rationale to the observed trends.     

Folding and stability of the three-stranded beta-sheet peptide Betanova: insights from molecular dynamics simulations

The dynamics of the three-stranded beta-sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20-ns simulations at 280 K indicate that the peptide remains very flexible under "folding" conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of "native"-like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptide's stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR-derived distance restraints and a possible mechanism for the folding of the peptide into the NMR-determined structure is proposed.