•Intrinsically disordered peptides (IDP) lack discernable tertiary structure and populate transient secondary structural elements.•IDP can play both physiological and pathological roles.•Simulations have played a critical role in determining structural ensembles of intrinsically disordered peptides.•Simulations have shed light into IDP function and self-assembly.Intrinsically disordered proteins (IDPs) and protein regions can facilitate a wide variety of complex physiological processes such as binding, signaling, and formation of membraneless organelles. They can however also play pathological roles by aggregating into cytotoxic oligomers and fibrils. Characterizing the structure and function of disordered proteins is an onerous task, primarily because these proteins adopt transient structures, which are difficult to capture in experiments. Simulations have emerged as a powerful tool for interpreting and augmenting experimental measurements of IDPs. In this review we focus on computer simulations of disordered protein structures, functions, assemblies, and emerging questions that, taken together, give an overview of the field as it exists today.

Translating sticky biological molecules—such as mussel foot proteins (MFPs)—into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue’s molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces.

The formation of a hydrophobic core is key to the folding and resulting function of most proteins in the cell. In several organisms, as well as in many in vitro experiments, protein folding is modulated by the presence of osmolytes, but the mechanism by which hydrophobic association occurs is not well understood. We present a study of the solvation thermodynamics of hydrophobic self-association in mixed-osmolyte urea–TMAO solutions, with neopentane as a model hydrophobic molecule. Using molecular dynamics simulations and the Kirkwood–Buff theory of solutions, we show that a sensitive balance between the TMAO–water and the TMAO–urea interactions governs the osmolyte-induced changes in hydrophobic association in mixed urea–TMAO solutions. This balance must be correctly incorporated in force-field parametrization because hydrophobic association can be either enhanced or prevented all together by slightly increasing or decreasing the osmolyte–water affinity and osmolyte–osmolyte self-affinity of TMAO molecules.

We investigate the relationship between the inherent secondary structure and aggregation propensity of peptides containing chameleon sequences (i.e., sequences that can adopt either α or β structure depending on context) using a combination of replica exchange molecular dynamics simulations, ion-mobility mass spectrometry, circular dichroism, and transmission electron microscopy. We focus on an eight-residue long chameleon sequence that can adopt an α-helical structure in the context of the iron-binding protein from Bacillus anthracis (PDB id 1JIG) and a β-strand in the context of the baculovirus P35 protein (PDB id 1P35). We show that the isolated chameleon sequence is intrinsically disordered, interconverting between α-helical and β-rich conformations. The inherent conformational plasticity of the sequence can be constrained by addition of flanking residues with a given secondary structure propensity. Intriguingly, we show that the chameleon sequence with helical flanking residues aggregates rapidly into fibrils, whereas the chameleon sequence with flanking residues that favor β-conformations has weak aggregation propensity. This work sheds new insights into the possible role of α-helical intermediates in fibril formation.

Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer’s disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a “superposition of ensembles” hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.

Protein-surface interactions are ubiquitous in both the cellular setting and in modern bioengineering devices, but how such interactions impact protein stability is not well understood. We investigate the folding of the GB1 hairpin peptide in the presence of self-assembled monolayers and graphite like surfaces using replica exchange molecular dynamics simulations. By varying surface hydrophobicity, and decoupling direct protein–surface interactions from water-mediated interactions, we show that surface wettability plays a surprisingly minor role in dictating protein stability. For both the β-hairpin GB1 and the helical miniprotein TrpCage, adsorption and stability is largely dictated by the nature of the direct chemical interactions between the protein and the surface. Independent of the surface hydrophobicity profile, strong protein–surface interactions destabilize the folded structure while weak interactions stabilize it.

Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; 273GKVQIINKKLDL284) and PHF6 (R3/wt; 306VQIVYKPVDLSK317) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*–PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function.

Trp-cage is an artificial miniprotein that is small, stable, and fast folding due to concerted hydrophobic shielding of a Trp residue by polyproline helices. Simulations have extensively characterized Trp-cage; however, the interactions of Trp-cage with organic surfaces (e.g., membranes) and their effect on protein conformation are largely unknown. To better understand these interactions we utilized a combination of replica-exchange molecular dynamics (REMD) and metadynamics (MetaD), to investigate Trp-cage folding on self-assembled monolayers (SAMs). We found that, with REMD and MetaD, Trp-cage strongly binds to neutral CH3 surfaces (−25kT) and moderately adsorbs to anionic COOH interfaces (−7.6kT), with hydrophobic interactions driving CH3 adhesion and electrostatic attractions driving COOH adhesion. Similar to solid-state surfaces, SAMs facilitate a number of intermediate Trp-cage conformations between folded and unfolded states. Regarding Trp-cage’s aromatic groups in neutral CH3 systems, Tyr becomes oriented parallel to the surface in order to maximize hydrophobic interactions while Trp remains caged perpendicular to the surface; however, Trp can reorient itself parallel to the interface as the miniprotein more closely binds to the surface. In contrast, Tyr and Trp are both repelled from COOH surfaces, though the Trp-cage still adheres to the anionic interface via Lys and its N-terminated Asn residue.

We present a novel hybrid MD-kMC algorithm that is capable of efficiently folding proteins in explicit solvent. We apply this algorithm to the folding of a small protein, Trp-Cage. Different kMC move sets that capture different possible rate limiting steps are implemented. The first uses secondary structure formation as a relevant rate event (a combination of dihedral rotations and hydrogen-bonding formation and breakage). The second uses tertiary structure formation events through formation of contacts via translational moves. Both methods fold the protein, but via different mechanisms and with different folding kinetics. The first method leads to folding via a structured helical state, with kinetics fit by a single exponential. The second method leads to folding via a collapsed loop, with kinetics poorly fit by single or double exponentials. In both cases, folding times are faster than experimentally reported values, The secondary and tertiary move sets are integrated in a third MD-kMC implementation, which now leads to folding of the protein via both pathways, with single and double-exponential fits to the rates, and to folding rates in good agreement with experimental values. The competition between secondary and tertiary structure leads to a longer search for the helix-rich intermediate in the case of the first pathway, and to the emergence of a kinetically trapped long-lived molten-globule collapsed state in the case of the second pathway. The algorithm presented not only captures experimentally observed folding intermediates and kinetics, but yields insights into the relative roles of local and global interactions in determining folding mechanisms and rates.

The aggregation of peptides on a lipid bilayer is studied using coarse-grained molecular dynamics in implicit solvent. Peptides bind to and self-assemble on the membrane surface into β-rich fibrillar aggregates, even under conditions where only disordered oligomers form in bulk solution. Relative to a solid surface, the membrane surface facilitates peptide mobility and a more complex network of morphology transitions as aggregation proceeds. Additionally, final aggregate structures realized on the membrane surface are distinct from those observed on a comparable solid surface. The aggregated fibrils alter the local structure and material properties of the lipid bilayer in their immediate vicinity but have only a modest effect on the overall bending rigidity of the bilayer.

The microtubule associated protein tau is essential for the development and maintenance of the nervous system. Tau dysfunction is associated with a class of diseases called tauopathies, in which tau is found in an aggregated form. This paper focuses on a small aggregating fragment of tau, 273GKVQIINKKLDL284, encompassing the (PHF6*) region that plays a central role in tau aggregation. Using a combination of simulations and experiments, we probe the self-assembly of this peptide, with an emphasis on characterizing the early steps of aggregation. Ion-mobility mass spectrometry experiments provide a size distribution of early oligomers, TEM studies provide a time course of aggregation, and enhanced sampling molecular dynamics simulations provide atomistically detailed structural information about this intrinsically disordered peptide. Our studies indicate that a point mutation, as well the addition of heparin, lead to a shift in the conformations populated by the earliest oligomers, affecting the kinetics of subsequent fibril formation as well as the morphology of the resulting aggregates. In particular, a mutant associated with a K280 deletion (a mutation that causes a heritable form of neurodegeneration/dementia in the context of full length tau) is seen to aggregate more readily than its wild-type counterpart. Simulations and experiment reveal that the ΔK280 mutant peptide adopts extended conformations to a greater extent than the wild-type peptide, facilitating aggregation through the pre-structuring of the peptide into a fibril-competent structure.

The structural properties of water molecules surrounding TMAO molecules are studied using a newly developed atomistic force field for TMAO, combined with a multiscale coarse-graining (MS-CG) force field derived from the atomistic simulations. The all-atom force field is parametrized to work with the OPLS force field and with SPC, TIP3P, and TIP4P water models. The dual-resolution modeling enables a complete study of the dynamical and structural properties of the system, with the CG model providing important new physical insights into which interactions are critical in determining the structure of water around TMAO. TMAO is an osmolyte that stabilizes protein structures under conditions of chemical, thermal, and pressure denaturation. This molecule is excluded from the surface of proteins, and its effect on protein stability is mediated through TMAO–water interactions. We find that TMAO strongly binds two to three water molecules and, surprisingly, that methyl groups repel both the other methyl groups of TMAO and water molecules alike. The latter result is important because it shows that methyl groups are not interacting with each other through the expected hydrophobic effect (which would be attractive and not repulsive) and that the repulsion of water molecules forces a clathrate-like hydrogen bond network around them. We speculate that TMAO is excluded from the vicinity of the protein because the peculiar structure of water around TMAO prevents this molecule from coming in close contact with the protein.

A two-component signal transduction pathway underlies the phenomenon of bacterial chemotaxis that allows bacteria to modulate their swimming behavior in response to environmental stimuli. The dimeric five-domain histidine kinase, CheA, plays a central role in the pathway, converting sensory signals to a chemical signal via trans-autophosphorylation between the P1 and P4 domains. This autophosphorylation is regulated via the networked interactions among the P5 domain of CheA, CheW, and chemoreceptors. Despite a wealth of structural information about these components and their interactions, the key question of how the kinase activity of the catalytic P4 domain is regulated by the signal received from the regulatory P5 domain remains poorly understood. We performed replica exchange molecular dynamics simulations on the CheA kinase core and found that while individual domains maintained their structural fold, these domains exhibited a variety of interdomain orientations due to two interdomain linkers. A partially populated conformation that adopts an interdomain arrangement is suitable for building a functional ternary complex. An allosteric network derived from this structural model implies critical roles for two linkers in CheA’s activity. The biochemical and biological functions of these linkers were assigned via a series of biochemical and genetic assays that show the P4–P5 linker controls the activation of CheA and the P3–P4 linker controls both the basal autophosphorylation activity and the activation of CheA. These results reveal the functional dependence between the two linkers and the essential role of the linkers in passing signal information from one domain to another.

Many problems of interest in modern science originate from the complex network of interactions of different molecular structures, each possessing its own typical length and time scale of relevance. In such materials, nontrivial properties emerge from the different length and time scales involved that could not be predicted from the properties of each individual subunit taken alone. A solution to the formidable theoretical and computational issues raised by these systems involves coarse-graining, a procedure in which multiple atoms are grouped into a few interaction sites. The coarse-grained approach aims at constructing an effective Hamiltonian from available information about the system and then using this Hamiltonian to investigate the behavior of the system on the length and time scales of interest. In this paper, we aim at determining how far we can coarse-grain a system using only the commonly used pairwise, spherically symmetric potentials, as well as assessing the impact of poor initial sampling on the quality of the resulting coarse-grained model. Coarse-graining is performed following the multiscale coarse-graining (MS-CG) methodology, and we use as a model system the N-methylacetamide (NMA) molecule, a simple representation of a peptide bond, which can adopt two conformations, cis and trans. Our simulations reveal that as the coarse-graining becomes more aggressive multibody effects start to emerge and that the initial sampling of conformations can adversely bias the model in the case of heavy coarse-graining.

For the last 20 years, a large volume of experimental and theoretical work has been undertaken to understand how chaperones like GroEL can assist protein folding in the cell. The most accepted explanation appears to be the simplest: GroEL, like most other chaperones, helps proteins fold by preventing aggregation. However, evidence suggests that, under some conditions, GroEL can play a more active role by accelerating protein folding. A large number of models have been proposed to explain how this could occur. Focused experiments have been designed and carried out using different protein substrates with conclusions that support many different mechanisms. In the current article, we attempt to see the forest through the trees. We review all suggested mechanisms for chaperonin-mediated folding and weigh the plausibility of each in light of what we now know about the most stringent, essential, GroEL-dependent protein substrates.

We present a driver program for performing replica-exchange molecular dynamics simulations with the Tinker package. Parallelization is based on the Message Passing Interface, with every replica assigned to a separate process. The algorithm is not communication intensive, which makes the program suitable for running even on loosely coupled cluster systems. Particular attention is paid to the practical aspects of analyzing the program output.Program summaryProgram title: TiReXCatalogue identifier: AEEK_v1_0Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEEK_v1_0.htmlProgram obtainable from: CPC Program Library, Queen's University, Belfast, N. IrelandLicensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.htmlNo. of lines in distributed program, including test data, etc.: 43 385No. of bytes in distributed program, including test data, etc.: 502 262Distribution format: tar.gzProgramming language: Fortran 90/95Computer: Most UNIX machinesOperating system: LinuxHas the code been vectorized or parallelized?: parallelized with MPIClassification: 16.13External routines: TINKER version 4.2 or 5.0, built as a libraryNature of problem: Replica-exchange molecular dynamics.Solution method: Each replica is assigned to a separate process; temperatures are swapped between replicas at regular time intervals.Running time: The sample run may take up to a few minutes.

We present a novel method for comparing the long-range part of force fields in the presence of a maximally cooperative network of nonbonded interactions. The method is based on mapping the potential energy surface of an infinite polypeptide chain in the gas phase by using cylindrical coordinates (the twist and pitch) as geometry descriptors. We apply our method to an infinite polyalanine chain and consider the AMBER99, AMBER99SB, CHARMM27, and OPLS-AA/L fixed partial-charge force fields and the protein-specific version of the AMOEBA polarizable force field. Results from our analysis are compared to those obtained from high-level density-functional theory (DFT) calculations. We find that all force fields produce stronger stabilization of the helical conformations as compared to DFT, with only AMBER99/AMBER99SB satisfactorily reproducing all three helical conformations (π, α, and 310).

The folding mechanisms of proteins are increasingly being probed through single-molecule experiments in which the protein is immobilized on a surface. Nevertheless, a clear understanding of how the surface might affect folding, and whether or not it changes folding from its bulk behavior, is lacking. In this work, we use molecular dynamics simulations of a model β-barrel protein tethered to a surface to systematically investigate how the surface impacts folding. In the bulk, this protein folds in a three-state manner through a compact intermediate state, and its transition state (TS) has a well formed hydrophobic core. Upon tethering, we find that folding rates and stability are impacted differently by the surface, with dependencies on both the length and location of the tether. Significant changes in folding times are observed for tether points that do not alter the folding temperature. Tethering also locally enhances the formation of structure for residues proximal to the tether point. We find that neither the folding mechanism nor the TS of this protein are altered if the tether is in a fully structured or completely unstructured region of the TS. By contrast, tethering in a partially structured region of the TS leads to dramatic changes. For one such tether point, the intermediate present in bulk folding is eliminated, leading to a two-state folding process with a heterogeneous, highly unstructured TS ensemble. These results have implications for both the design of single-molecule experiments and biotechnological applications of tethered proteins.

Recent experiments suggest that the folding of certain proteins can take place entirely within a chaperonin-like cavity. These substrate proteins experience folding rate enhancements without undergoing multiple rounds of ATP-induced binding and release from the chaperonin. Rather, they undergo only a single binding event, followed by sequestration into the chaperonin cage. The present work uses molecular dynamics simulations to investigate the folding of a highly frustrated protein within this chaperonin cavity. The chaperonin interior is modeled by a sphere with a lining of tunable degree of hydrophobicity. We demonstrate that a moderately hydrophobic environment, similar to the interior of the GroEL cavity upon complexion with ATP and GroES, is sufficient to accelerate the folding of a frustrated protein by more than an order of magnitude. Our simulations support a mechanism by which the moderately hydrophobic chaperonin environment provides an alternate pathway to the native state through a transiently bound intermediate state.

The mechanism and thermodynamics of folding of the Src homology 3 (SH3) protein domain are characterized at an atomic level through molecular dynamics with importance sampling. This methodology enables the construction of the folding free energy landscape of the protein as a function of representative reaction coordinates. We observe that folding proceeds in a downhill manner under native conditions, with early compaction and structure formation in the hydrophobic sheet consisting of the three central β strands of the protein. This state bears considerable resemblance to the experimentally determined transition state for folding. Folding proceeds further with the formation of the second hydrophobic sheet consisting of the terminal strands and the RT loop. The final stages of folding appear to involve the formation of the hydrophobic core through the expulsion of water molecules bridging the two hydrophobic sheets. This work sheds new light on the complementary roles of sequence and topology in governing the folding mechanism of small proteins and provides further support for the role of water in facilitating the late stages in folding.

The conformational states sampled by the Alzheimer amyloid β (10-35) (Aβ 10-35) peptide were probed using replica-exchange molecular dynamics (REMD) simulations in explicit solvent. The Aβ 10-35 peptide is a fragment of the full-length Aβ 40/42 peptide that possesses many of the amyloidogenic properties of its full-length counterpart. Under physiological temperature and pressure, our simulations reveal that the Aβ 10-35 peptide does not possess a single unique folded state. Rather, this peptide exists as a mixture of collapsed globular states that remain in rapid dynamic equilibrium with each other. This conformational ensemble is dominated by random coil and bend structures with insignificant presence of an α-helical or β-sheet structure. The 3D structure of Aβ 10-35 is seen to be defined by a salt bridge formed between the side-chains of K28 and D23. This salt bridge is also observed in Aβ fibrils and our simulations suggest that monomeric conformations of Aβ 10-35 contain pre-folded structural motifs that promote rapid aggregation of this peptide.

The amyloid-β(25–35) peptide plays a key role in the etiology of Alzheimer's disease due to its extreme toxicity even in the absence of aging. Because of its high tendency to aggregate and its low solubility in water, the structure of this peptide is still unknown. In this work, we sought to understand the early stages of aggregation of the amyloid-β(25–35) peptide by conducting simulations of oligomers ranging from monomers to tetramers. Our simulations show that although the monomer preferentially adopts a β-hairpin conformation, larger aggregates have extended structures, and a clear transition from compact β-hairpin conformations to extended β-strand structures occurs between dimers and trimers. Even though β-hairpins are not present in the final architecture of the fibril, our simulations indicate that they play a critical role in fibril growth. Our simulations also show that β-sheet structures are stabilized when a β-hairpin is present at the edge of the sheet. The binding of the hairpin to the sheet leads to a subsequent destabilization of the hairpin, with part of the hairpin backbone dangling in solution. This free section of the peptide can then recruit an extra monomer from solution, leading to further sheet extension. Our simulations indicate that the peptide must possess sufficient conformational flexibility to switch between a hairpin and an extended conformation in order for β-sheet extension to occur, and offer a rationalization for the experimental observation that overstabilizing a hairpin conformation in the monomeric state (for example, through chemical cross-linking) significantly hampers the fibrillization process.

Congo red (CR) is a commonly used histological amyloid dye and a weak amyloid inhibitor. There is currently no experimentally available structure of CR bound to an amyloid fibril and the binding modes, and the mechanisms governing its inhibitory and optical properties are poorly understood. In this work, we present the first, to our knowledge, atomistically detailed picture of CR binding to protofibrils of the Alzheimer Aβ9–40 peptide. We identify three major binding modes, with the primary mode residing in the grooves formed by the β-sheets, and observe a restriction of the torsional rotation of the CR molecule upon binding. Our simulations reveal a novel, to our knowledge, electrostatic steering mechanism that plays an important role in the initial recognition and binding of CR to the positively charged surface residues of the fibril. Our simulations provide new, to our knowledge, insights into the striking spectrophotometric and inhibitory properties of CR. In particular, we show that birefringence upon CR binding is due to the anisotropic orientation of the CR dipoles resulting from the spatial ordering of these molecules in the grooves along the fibril axis. The fluorescent enhancement of the bound CR, in turn, is associated with the torsional restriction of this molecule upon binding.

The self-assembly of the KFFE peptide was studied using replica exchange molecular dynamics simulations with a fully atomic description of the peptide and explicit solvent. The relative roles of the aromatic residues and oppositely charged end groups in stabilizing the earliest oligomers and the end-products of aggregation were investigated. β and non-β-peptide conformations compete in the monomeric state as a result of a balancing between the high β-sheet propensity of the phenylalanine residues and charge-charge interactions that favor non-β-conformations. Dimers are present in β- and non-β-sheet conformations and are stabilized primarily by direct and water-mediated charge-charge interactions between oppositely charged side chains and between oppositely charged termini, with forces between aromatic residues playing a minor role. Dimerization to a β-sheet, fibril-competent state, is seen to be a cooperative process, with the association process inducing β-structure in otherwise non-β-monomers. We propose a model for the KFFE fibril, with mixed interface and antiparallel sheet and strand arrangements, which is consistent with experimental electron microscopy measurements. Both aromatic and charge-charge interactions contribute to the fibril stability, although the dominant contribution arises from electrostatic interactions.

The C-terminus of amyloid β-protein (Aβ) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Aβ42 have been shown to disrupt oligomerization and to strongly inhibit Aβ42-induced neurotoxicity. Here we study the structures of selected CTFs [Aβ(x–42); x = 29–31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable β-structure: β-hairpin for Aβ(x–42) (x = 29–31) and extended β-strand for Aβ(39–42). The β-hairpin of Aβ(30–42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the β-hairpin in Aβ42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of β-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ9-40 protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ17-42 revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ17-42 cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.

Natively disordered proteins belong to a unique class of biomolecules whose function is related to their flexibility and their ability to adopt desired conformations upon binding to substrates. In some cases these proteins can bind multiple partners, which can lead to distinct structures and promiscuity in functions. In other words, the capacity to recognize molecular patterns on the substrate is often essential for the folding and function of intrinsically disordered proteins. Biomolecular pattern recognition is extremely relevant both in vivo (e.g., for oligomerization, immune response, induced folding, substrate binding, and molecular switches) and in vitro (e.g., for biosensing, catalysis, chromatography, and implantation). Here, we use a minimalist computational model system to investigate how polar/nonpolar patterns on a surface can induce the folding of an otherwise unstructured peptide. We show that a model peptide that exists in the bulk as a molten globular state consisting of many interconverting structures can fold into either a helix-coil-helix or an extended helix structure in the presence of a complementary designed patterned surface at low hydrophobicity (3.7%) or a uniform surface at high hydrophobicity (50%). However, we find that a carefully chosen surface pattern can bind to and catalyze the folding of a natively unfolded protein much more readily or effectively than a surface with a noncomplementary or uniform distribution of hydrophobic residues.

Thioflavin T (ThT) is a fluorescent dye commonly used to stain amyloid plaques, but the binding sites of this dye onto fibrils are poorly characterized. We present molecular dynamics simulations of the binding of ThT and its neutral analog BTA-1 [2-(4′-methylaminophenyl)benzothiazole] to model protofibrils of the Alzheimer's disease Aβ16–22 (amyloid β) peptide. Our simulations reveal two binding modes located at the grooves of the β-sheet surfaces and at the ends of the β-sheet. These simulations provide new insight into recent experimental work and allow us to characterize the high-capacity, micromolar-affinity site seen in experiment as binding to the β-sheet surface grooves and the low-capacity, nanomolar-affinity site seen as binding to the β-sheet extremities of the fibril. The structure–activity relationship upon mutating charged ThT to neutral BTA-1 in terms of increased lipophilicity and binding affinity was studied, with calculated solvation free energies and binding energies found to be in qualitative agreement with the experimental measurements.

The GroEL chaperonin has the ability to behave as an unfoldase, repeatedly denaturing proteins upon binding, which in turn can free them from kinetic traps and increase their folding rates. The complex formed by GroEL+GroES+ATP can also act as an infinite dilution cage, enclosing proteins within a protective container where they can fold without danger of aggregation. Controversy remains over which of these two properties is more critical to the GroEL/ES chaperonin's function. We probe the importance of the unfoldase nature of GroEL under conditions where aggregation is the predominant protein degradation pathway. We consider the effect of a hypothetical mutation to GroEL which increases the cycle frequency of GroEL/ES by increasing the rate of hydrolysis of GroEL-bound ATP. Using a simple kinetic model, we show that this modified chaperonin would be self-defeating: any potential reduction in folding time would be negated by an increase in time spent in the bulk, causing an increase in aggregation and a net decrease in protein folding yields.

Although the amyloid dye thioflavin-T (ThT) is among the most widely used tools in the study of amyloid fibrils, the mechanism by which ThT binds to fibrils and other β-rich peptide self-assemblies remains elusive. The development of the water-soluble peptide self-assembly mimic (PSAM) system has provided a set of ideal model proteins for experimentally exploring the properties and minimal dye-binding requirements of amyloid fibrils. PSAMs consist of a single-layer β-sheet (SLB) capped by two globular domains, which capture the flat, extended β-sheet features common among fibril-like surfaces. Recently, a PSAM that binds to ThT with amyloid-like affinity (low micromolar Kd) has been designed, and its crystal structure in the absence of bound ThT was determined. This PSAM thus provides a unique opportunity to examine the interactions of ThT with a β-rich structure. Here, we present molecular dynamics simulations of the binding of ThT to this PSAM β-sheet. We show that the primary binding site for ThT is along a shallow groove formed by adjacent Tyr and Leu residues on the β-sheet surface. These simulations provide an atomic-scale rationale for this PSAM's experimentally determined dye-binding properties. Together, our results suggest that an aromatic–hydrophobic groove spanning across four consecutive β-strands represents a minimal ThT binding site on amyloid fibrils. Grooves formed by aromatic–hydrophobic residues on amyloid fibril surfaces may therefore offer a generic mode of recognition for amyloid dyes.

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21–30 fragment of the Alzheimer amyloid β-protein (Aβ) is investigated by replica-exchange molecular dynamics simulations. The 21–30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Aβ. Our simulations reveal that the 24–28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Aβ. Enhanced aggregation in Aβ with familial Alzheimer's disease substitutions may result from the depletion of the E22–K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21–30 segment that can impact the aggregation of Aβ. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Aβ folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Aβ are fundamentally different.

The microtubule associated protein tau is essential for the development and maintenance of the nervous system. Tau dysfunction is associated with a class of diseases called tauopathies, in which tau is found in an aggregated form. This paper focuses on a small aggregating fragment of tau, 273GKVQIINKKLDL284, encompassing the (PHF6*) region that plays a central role in tau aggregation. Using a combination of simulations and experiments, we probe the self-assembly of this peptide, with an emphasis on characterizing the early steps of aggregation. Ion-mobility mass spectrometry experiments provide a size distribution of early oligomers, TEM studies provide a time course of aggregation, and enhanced sampling molecular dynamics simulations provide atomistically detailed structural information about this intrinsically disordered peptide. Our studies indicate that a point mutation, as well the addition of heparin, lead to a shift in the conformations populated by the earliest oligomers, affecting the kinetics of subsequent fibril formation as well as the morphology of the resulting aggregates. In particular, a mutant associated with a K280 deletion (a mutation that causes a heritable form of neurodegeneration/dementia in the context of full length tau) is seen to aggregate more readily than its wild-type counterpart. Simulations and experiment reveal that the ΔK280 mutant peptide adopts extended conformations to a greater extent than the wild-type peptide, facilitating aggregation through the pre-structuring of the peptide into a fibril-competent structure.