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Amyloid-beta (Aβ) toxic oligomers are critical early players in the molecular pathology of Alzheimer's disease (AD). We have developed a Soluble Oligomer Binding Assay (SOBA-AD) for detection of these Aβ oligomers that contain α-sheet secondary structure that discriminates plasma samples from patients on the AD continuum from non-AD controls. We tested 265 plasma samples from two independent cohorts to investigate the performance of SOBA-AD. Testing was performed at two different sites, with different personnel, reagents, and instrumentation. Across two cohorts, SOBA-AD discriminated AD patients from cognitively unimpaired (CU) subjects with 100% sensitivity, > 95% specificity, and > 98% area under the curve (AUC) (95% CI 0.95–1.00). A SOBA-AD positive readout, reflecting α-sheet toxic oligomer burden, was found in AD patients, and not in controls, providing separation of the two populations, aside from 5 SOBA-AD positive controls. Based on an earlier SOBA-AD study, the Aβ oligomers detected in these CU subjects may represent preclinical cases of AD. The results presented here support the value of SOBA-AD as a promising blood-based tool for the detection and confirmation of AD. [ABSTRACT FROM AUTHOR]

Abstract Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial–temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding. Graphical abstract Unlabelled Image Highlights • Examination of protein folding and unfolding pathways via several different methods scaling from the atomistic level to whole-structure and multi-structure levels • Visualizing folding in multiple ways provides a more comprehensive picture of specific interactions that lead to different conformational states. [ABSTRACT FROM AUTHOR]

Abstract Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure—α-sheet. De novo designed peptides with alternating l - and d -amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans , α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation. Graphical abstract Unlabelled Image Highlights • Streptococcus mutans has recently been identified to form functional amyloid, but little is known about the mechanism of amyloid fibril formation. Comparing the effects of α-sheet peptides in S. mutans and other amyloid systems sheds light on the intermediates present during fibril formation. • Multiple α-sheet peptides inhibit amyloid fibril formation in S. mutans biofilms and in bacteria present in whole human saliva samples compared to β-sheet and random coil control peptides. Inhibition occurred in biofilms grown in a plate and in bacterial accumulations on tooth-like hydroxyapatite surfaces. • TEM imaging reveals that fibril structures present in S. mutans biofilms are no longer present upon the addition of α-sheet peptides. • These results suggest that an α-sheet intermediate is involved in amyloid fibril formation in S. mutans biofilms. [ABSTRACT FROM AUTHOR]

Colocalization of microbial pathogens and the β-amyloid peptide (Aβ) in the brain of Alzheimer’s disease (AD) patients suggests that microbial infection may play a role in sporadic AD. Aβ exhibits antimicrobial activity against numerous pathogens, supporting a potential role for Aβ in the innate immune response. While mammalian amyloid is associated with disease, many bacteria form amyloid fibrils to fortify the biofilm that protects the cells from the surrounding environment. In the microbial AD hypothesis, Aβ aggregates in response to infection to combat the pathogen. We hypothesize that this occurs through toxic Aβ oligomers that contain α-sheet structure and form prior to fibrillization. De novo designed α-sheet peptides specifically bind to the α-sheet structure present in the oligomers of both bacterial and mammalian amyloidogenic proteins to neutralize toxicity and inhibit aggregation. Here, we measure the effect of E. coli on Aβ, including upregulation, aggregation, and toxicity. Additionally, we determined the effect of Aβ structure on E. coli amyloid fibrils, or curli comprised of the CsgA protein, and biofilm formation. We found that curli formation by E. coli increased Aβ oligomer production, and Aβ oligomers inhibited curli biogenesis and reduced biofilm cell density. Further, curli and biofilm inhibition by Aβ oligomers increased E. coli susceptibility to gentamicin. Toxic oligomers of Aβ and CsgA interact via α-sheet interactions, neutralizing their toxicity. These results suggest that exposure to toxic oligomers formed by microbial pathogens triggers Aβ oligomer upregulation and aggregation to combat infection via selective interactions between α-sheet oligomers to neutralize toxicity of both species with subsequent inhibition of fibrillization. [ABSTRACT FROM AUTHOR]

Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is a-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process. [ABSTRACT FROM AUTHOR]

Abstract: Molecular dynamics simulations are a powerful theoretical tool to model the protein folding process in atomistic details under realistic conditions. Combined with a number of experimental techniques, simulations provide a detailed picture of how a protein folds or unfolds in the presence of explicit solvent and other molecular species, such as cosolvents, osmolytes, cofactors, active binding partners or inert crowding agents. The denaturing effects of temperature, pressure and external mechanical forces can also be probed. Qualitative and quantitative agreement with experiment contributes to a comprehensive molecular picture of protein states along the folding/unfolding pathway. The variety of systems examined reveals key features of the protein folding process. [Copyright &y& Elsevier]

Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our consensus domain dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1,695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predictions suggest that 40% of proteins have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. [ABSTRACT FROM AUTHOR]

High performance computing is leading to unprecedented volumes of data. Relational databases offer a robust and scalable model for storing and analyzing scientific data. However, these features do not come without a cost-significant design effort is required to build a functional and efficient repository. Modeling protein simulation data in a relational database presents several challenges: The data captured from individual simulations are large, multidimensional, and must integrate with both simulation software and external data sites. Here, we present the dimensional design and relational implementation of a comprehensive data warehouse for storing and analyzing molecular dynamics simulations using SQL Server. [ABSTRACT FROM AUTHOR]

Graphs are rapidly becoming a powerful and ubiquitous tool for the analysis of protein structure and for event detection in dynamical protein systems. Despite their rise in popularity, however, the graph representations employed to date have shared certain features and parameters that have not been thoroughly investigated. Here, we examine and compare variations on the construction of graph nodes and graph edges. We propose a graph representation based on chemical groups of similar atoms within a protein rather than residues or secondary structure and find that even very simple analyses using this representation form a powerful event detection system with significant advantages over residue-based graph representations. We additionally compare graph edges based on probability of contact to graph edges based on contact strength and analyses of the entire graph structure to an alternative and more computationally tractable node-based analysis. We develop the simplest useful technique for analyzing protein simulations based on these comparisons and use it to shed light on the speed with which static protein structures adjust to a solvated environment at room temperature in simulation. [ABSTRACT FROM AUTHOR]

Molecular dynamics (MD) is the only technique availablefor obtainingdynamic protein data at atomic spatial resolution and picosecond orfiner temporal resolution. In recent years, the cost of computationalresources has decreased exponentially while the number of known proteinstructures, many of which are not characterized biochemically, hasincreased rapidly. These events have led to an increase in the useof MD in biological research, both to examine phenomena that cannotbe resolved experimentally and to generate hypotheses that directfurther experimental research. In fact, several databases of MD simulationshave arisen in recent years. MD simulations, and especially MD simulationdatabases, contain massive amounts of data, yet interesting phenomenaoften occur over very short time periods and on the scale of onlya few atoms. Analysis of such data must balance these fine-detailevents with the global picture they create. Here, we address the multiscalenature of the problem by comparing several MD analysis methods toshow their strengths and weaknesses at various scales using the wild-typeand R282W mutant forms of the DNA-binding domain of protein p53. Byleveraging these techniques together, we are able to pinpoint fine-detailand big picture differences between the protein’s variants.Our analyses indicate that the R282W mutation of p53 destabilizesthe L1 loop and loosens the H2 helix conformation, but the loosenedL1 loop can be rescued by residue H115, preventing the R282W mutationfrom completely destabilizing the protein or abolishing activity. [ABSTRACT FROM AUTHOR]

As high-throughput molecular dynamics simulations of proteins become more common and the databases housing the results become larger and more prevalent, more sophisticated methods to quickly and accurately mine large numbers of trajectories for relevant information will have to be developed. One such method, which is only recently gaining popularity in molecular biology, is the continuous wavelet transform, which is especially well-suited for time course data such as molecular dynamics simulations. We describe techniques for the calculation and analysis of wavelet transforms of molecular dynamics trajectories in detail and present examples of how these techniques can be useful in data mining. We demonstrate that wavelets are sensitive to structural rearrangements in proteins and that they can be used to quickly detect physically relevant events. Finally, as an example of the use of this approach, we show how wavelet data mining has led to a novel hypothesis related to the mechanism of the protein γδ resolvase. [ABSTRACT FROM AUTHOR]

The prion diseases are a class of neurodegenerative diseases caused by the misfolding and aggregation of the prion protein (PrPC) into toxic and infectious oligomers (PrPSc). These oligomers are critical to understanding and combating these diseases. Differences in the sequence of PrP affect disease susceptibility, likely by shifting the tolerance of the protein for adaptation to PrPSc conformations and/or the recognition event between PrPSc and PrPC prior to conversion of the PrPC. We selected two sets of PrPSc-resistant mutant sequences for solvated atomistic molecular dynamics simulation to investigate the structural basis of resistance. The first group involved mutation in the X-loop (residues 164-171) resulting from selective breeding of sheep. The second group included eight mutants in mice identified by random mutagenesis targeting helix C followed by screening in cell cultures. Multiple simulations were performed of 14 different mutant and control constructs under different pH conditions for a total of 3.6 μs of simulation time. The X-loop formed a stable turn at neutral pH in wild-type PrP from both species. PrPSc-resistant mutations disrupted this turn even though only one of the mutants is in the X-loop. The X-loop is compact and buried in our previously described spiral models of PrPSc-like oligomers. On the basis of the findings presented here and in the context of the spiral oligomer model, we propose that expansion of the X-loop disrupts protofibril packing, providing a structural basis for resistance. [ABSTRACT FROM PUBLISHER]

Abstract: Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior. [Copyright &y& Elsevier]

The p53 tumor suppressor is a transcription factor involved in many important signaling pathways, such as apoptosis and cell-cycle arrest. In over half of human cancers, p53 function is compromised by a mutation in its gene. Mutations in the p53 DNA-binding core domain destabilize the structure and reduce DNA.binding activity. We performed molecular dynamics simulations at physiological temperature to study the structural and dynamic effects of the L145Q V157F, and R282W cancer-associated mutations in comparison to the wild-type protein. While there were common regions of destabilization in the mutant simulations, structural changes particular to individual mutations were also observed. Significant backbone deviations of the H2 helix and S7-S8 loop were observed in all mutant simulations; the H2 helix binds to DNA In addition, the L14SQ and V1S7F mutations, which are located in the β-sandwich core of the domain, disrupted the β-sheet structure and the loop-sheet-helix motif The R282W mutation caused distortion of the loop-sheet-helix motif but otherwise this mutant was similar to the wild-type structure. The introduction of these mutations caused rearrangement of the DNA-binding surface, consistent with their reduced DNA-binding activity. The simulations reveal detailed effects of the mutations on the stability and dynamics of p53 that may provide insight for therapeutic approaches. [ABSTRACT FROM AUTHOR]

The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. [ABSTRACT FROM AUTHOR]

Abstract: Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases. [ABSTRACT FROM AUTHOR]

Methyltransferases catalyze the methylation processes essential for protein/DNA repair, transcriptional regulation, and drug metabolism in vivo. More than 500 human methyltransferase polymorphisms have been identified, many of which are linked to disease. We mapped all available coding polymorphisms of seven methyltransferases onto their structures to address their structural significance, and identified a polymorphic hotspot ∼20Å from the active site in four of the proteins. Molecular dynamics simulations of these proteins reveal a common mechanism of destabilization: the mutations alter important side-chain contacts within the polymorphic site that are propagated through the protein, thereby distorting the active site. We propose that this hotspot might have arisen to modulate enzymatic activity, with decreased activity actually conferring an advantage in three of the four methyltransferases. [ABSTRACT FROM AUTHOR]

It has been suggested that denatured proteins are predisposed toward the left-handed polyproline II (PII) conformation. One possible source of PII stability in the denatured state is water bridges. Water bridges are networks of water molecules that link nearby hydrogen bond acceptors and/or donors on proteins. On the basis of the proposed behavior of PII and water bridges, the propensity of a residue to participate in water bridges should be correlated with its PII propensity. To test this hypothesis, we analyzed the following data sets: 2351 high-resolution crystal structures, and the native and denatured states of 188 different proteins from all-atom, explicit-solvent molecular dynamics (MD) simulations, which are part of our Dynameomics effort. We found that water bridges do not explain the high frequency of PII in denatured states; such bridges are less frequent around PII than around other conformations. Thus, this analysis casts doubt on water bridges as a dominant factor determining the residue-based PII propensities. [ABSTRACT FROM PUBLISHER]

Protein L-isoaspartate O-methyltransferase (PIMT) repairs isoaspartate residues in damaged proteins, and it contains a Val–Ile polymorphismin in α5, ∼13 Å from its active site. Val119 has lower activity and thermal stability but increased affinity for endogenous substrates. Studies suggest that heterozygosity for Val/Ile favors efficient isoaspartate repair. We have performed multiple molecular dynamics simulations of 119I and 119V PIMT. Both V119 and I119 interact with the same residues throughout all of the simulations. However, the larger Ile altered the orientations of α5 and β5, both of which have co-substrate binding residues on their distal ends. I119 increases the flexibility of several residues, loosening up the S-adenosylmethionine (SAM)-binding site. These subtle changes are propagated towards the isoaspartate-docking site via residues common to both active sites. The increased mobility in 119I PIMT reorients α3, resulting in a salt-bridge network at the substrate-binding interface that disrupts several key side-chain interactions in the isoaspartate site. In contrast, 119V PIMT remains quite rigid with little change to the co-substrate binding site, which could hinder SAM's binding and release, accounting for the decreased activity. These results shed light on the molecular basis behind the decreased activity and increased specificity for endogenous substrates of 119V PIMT relative to the 119I variant. 119I PIMT catalyzes the methylation reaction but may have difficulties recognizing and orienting specific substrates due to its distorted substrate-binding site. Heterozygosity for both the Ile and Val alleles may provide the best of both worlds, allowing the fast and specific methylation of damaged proteins. [ABSTRACT FROM PUBLISHER]

Prion diseases, in which the conformational transition of the native prion protein (PrP) to a misfolded form causes aggregation and subsequent neurodegeneration, have fascinated the scientific community as this transmissible disease appears to be purely protein-based. Disease can arise due to genetic factors only. At least 30 single point mutations have been indicated to cause disease in humans. Somehow, these mutations must influence the stability, processing and/or cellular interactions of PrP, such that aggregation can occur and disease develops. In this review, the current evidence for such effects of single point mutations is discussed, indicating that PrP can be affected in many different ways, although questions remain about the mechanism by which mutations cause disease. [ABSTRACT FROM PUBLISHER]

Human catechol O-methyltransferase (COMT) contains three common polymorphisms (A22S, AS2T, and VIOSM), two of which (A22S and V108M) render the protein susceptible to deactivation by temperature or oxidation. We have performed multiple molecular dynamics simulations of the wild-type, A22S, A52T, and V108M COMT proteins to explore the structural consequences of these mutations In total, we have amassed more than 1.4μs of simulation time, representing the largest set of simulations detailing the feets of polymorphisms on a protein system to date The A52T mutation had no significant effect on COMT structure in accord with experiment, thereby serving as a good negative control for the simulation set Residues 22 (α2) and 108 (α5) interact with each other throughout the simulations and are located in a polymorphic hotspot ∼20 A from the active site. Introduction of either the larger Ser (22) or Met (108) tightens this interaction, pulling α2 and α5 toward each other and away from the protein core. The V108M polymorphism rearranges active-site residues in α5, β3, and β6, increasing the S-adenosylmethionine site solvent exposure. The A22S mutation reorients α2, moving critical carechol-binding residues away from the substrate-binding pocket The A22S and V108M polymorphisms evolved independently in Northern European and Asian population. While the decreased activities of both A22S and V108M COMT are associated with an increases risk for schizophrenia, the V108M-induced destabilization is also linked with improved cognitive function. These results suggest that polymorphisms within this hotspot may have evolved to regulate COMT activity and that heterozygosity for either mutation may be advantageous. [ABSTRACT FROM AUTHOR]

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo, there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrPC) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrPC as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrPC, but they do appreciably modify the accessibility of the polypeptide surface PrPC. In addition, the simulations of membrane-bound PrPC revealed likely recognition domains for the disease-initiating PrPC:PrPSc (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species. [ABSTRACT FROM AUTHOR]

Abstract: Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles [TPMT*2 (A80P), *3A (A154T/Y240C), *3B (A154T) and *3C (Y240C)] to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT*2 disrupts helix α3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding site. The larger threonine alters the packing of substrate-binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in β-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix α8 are lost in TPMT*3C. Residues 154 and 240 in TPMT*3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations. [Copyright &y& Elsevier]

The misfolding and aggregation of the prion protein (PrP) is the primary cause of a group of infectious neurodegenerative diseases including Creutzfeldt-Jacob disease in humans and Bovine Spongiform Encephalopathy in cows. A single disease can exhibit different infectious strains distinguishable by incubation time and morphology or distribution of the aggregates. Infected brain tissue from one species can be used to infect other species, but with different efficiencies, suggesting a spectrum of species compatibility. If PrP is, as widely believed, the sole component of infection, then the species and strain differences must be accounted for by the structure of the aggregates, likely influenced by each species’ PrP sequence. As there are no high-resolution data exploring this hypothesis, we performed molecular dynamics simulations of PrP for human, bovine, hamster, and D147N mutant hamster sequences at low pH to induce misfolding of the protein. We selected representative converted structures from each of the four sequences and, with the guidance of experimental data, constructed models of the infectious aggregates. Both hamster monomers showed high flexibility during conversion, suggesting hamster may more easily adopt altered conformations, which in turn may explain why it is more easily infected by some other species. Human and bovine aggregates were similar, with monomers docking in P31 symmetry to form a left-handed spiral. In contrast, hamster aggregates formed a P31 right-handed spiral. We detail the differences in the converted monomers that give rise to this difference and show that our results compare favorably with experimental data. [ABSTRACT FROM AUTHOR]

Conformational changes in the prion protein cause transmissible spongiform encephalopathies, also referred to as prion diseases. In its native state, the prion protein is innocuous (PrPC), but it can misfold into a neurotoxic and infectious isoform (PrPSc). The full-length cellular form of the prion protein consists of residues 23–230, with over half of the sequence belonging to the unstructured N-terminal domain and the remaining residues forming a small globular domain. During misfolding and aggregation, portions of both the structured and unstructured domains are incorporated into the aggregates. After limited proteolysis by proteinase K, the most abundant fragment from brain-derived prion fibrils is a 141-residue fragment composed of residues 90–230. Here we describe simulations of this fragment of the human prion protein at low pH, which triggers misfolding, and at neutral pH as a control. The simulations, in agreement with experiment, show that this biologically and pathologically relevant prion construct is stable and native-like at neutral pH. In contrast, at low pH the prion protein is destabilized via disruption of critical long-range salt bridges. In one of the low pH simulations this destabilization resulted in a conformational transition to a PrPSc-like isoform consistent with our previous simulations of a smaller construct. [ABSTRACT FROM AUTHOR]

Abstract: Both folded and unfolded conformations should be observed for a protein at its melting temperature (T m), where ΔG between these states is zero. In an all-atom molecular dynamics simulation of chymotrypsin inhibitor 2 (CI2) at its experimental T m, the protein rapidly loses its low-temperature native structure; it then unfolds before refolding to a stable, native-like conformation. The initial unfolding follows the unfolding pathway described previously for higher-temperature simulations: the hydrophobic core is disrupted, the β-sheet pulls apart and the α-helix unravels. The unfolded state reached under these conditions maintains a kernel of structure in the form of a non-native hydrophobic cluster. Refolding simply reverses this path, the side-chain interactions shift, the helix refolds, and the native packing and hydrogen bonds are recovered. The end result of this refolding is not the initial crystal structure; it contains the proper topology and the majority of the native contacts, but the structure is expanded and the contacts are long. We believe this to be the native state at elevated temperature, and the change in volume and contact lengths is consistent with experimental studies of other native proteins at elevated temperature and the chemical denaturant equivalent of T m. [Copyright &y& Elsevier]

The problem of how a protein folds from a linear chain of amino acids to the three-dimensional structure necessary for function is often investigated using proteins with a low degree of sequence identity that adopt different folds. The design of pairs of proteins with a high degree of sequence identity but different folds offers the opportunity for a complementary study; in two highly similar sequences, which residues are the most important in directing folding to a particular structure? Here we use molecular dynamics simulations to characterize the folding-unfolding pathways of a pair of proteins designed by Bryan and co-workers [Alexander, P. A., et al. (2005) Biochemistry 44, 14045-14054; He, Y. N., et al. (2005) Biochemistry 44, 14055-14061]. Despite being 59% identical, the two protein sequences fold to two different structures. The first sequence folds to the α+β protein G structure and the second to the all-α-helical protein A structure. We show that the final protein structure is determined early along the folding pathway. In folding to the protein G structure, the single α-helix (α1) and the β3-β4 turn fold early. Formation of the hairpin turn essentially prevents folding to helical structure in this region of the protein. This early structure is then consolidated by formation of long-range hydrophobic interactions between al and the β3-β4 turn. The protein A sequence differs both in the residues that form the β3-β4 turn and also in many of the residues that form the early hydrophobic interactions in the protein G structure. Instead, in the protein A sequence, a more hierarchical mechanism is observed, with helices folding before many of the tertiary interactions are formed. We find that small, but critical, sequence differences determine the topology of the protein early along the folding pathway, which help to explain the process by which one fold can evolve into another. [ABSTRACT FROM AUTHOR]

The article presents the molecular dynamics (MD) simulations of proteins in solution to examine the detailed processes of protein unfolding in the atomic-level. It is likewise employed to elucidate the kinetic pathways and these simulations are performed at high temperature to overcome energetic barriers to unfolding. The understanding of protein folding/unfolding is relevant for biological processes like aging, human diseases, protein degradation and protein translocation.

The self-assembly of β2-microglobuhn into fibrils leads to dialysis-related amyloidosis. pH- mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self- assembles into amyloid fibrils. Two partially folded intermediates of β2-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of a-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual a-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of a-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates. [ABSTRACT FROM AUTHOR]

Abstract: Prion diseases are neurodegenerative diseases causally linked to the partial unfolding and subsequent misfolding and aggregation of the prion protein (PrP). While most proteins fold into a single low energy state, PrP can fold into two distinct isoforms. In its innocuous state, denoted as PrPC, the protein has predominantly α-helical secondary structure, however, PrPC can misfold into an isoform rich in extended structure capable of forming toxic and infectious aggregates. While prion disease is believed to be a protein-only disease, one not requiring any non-protein elements for propagation, the different environments the protein finds itself in vivo likely influence its ability to misfold and aggregate. In this review we will examine various molecules, covalent modifications and environments PrP faces in vivo and the effect they have on PrP''s local environment and, potentially, conformation. Included in this discussion are: (1) pH, (2) carbohydrates, (3) lipid membranes, (4) metal ions, and (5) small molecules. To cite this article: M.L. DeMarco, V. Daggett, C. R. Biologies 328 (2005). [Copyright &y& Elsevier]

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

Bovine pancreatic ribonuclease (RNase A) deserves a special place among the numerous proteins that form oligomers by three-dimensional domain swapping. In fact, under destabilizing conditions and at high protein concentrations, it can swap two different domains, the N-terminal a-helix or the C-terminal β-strand, leading to dimers with different quaternary structures. With the change in the unfolding conditions, the relative abundance of the two dimers varies, and the prevalence of one dimer over the other is inverted. To investigate the dynamic behavior of the termini, four independent 10 ns high-temperature molecular dynamics simulations of RNase A were carried out at two different pH values in an attempt to reproduce the experimental conditions of neutral and very low pH that favor the formation of the N- and C-terminal domain-swapped dimers, respectively. In agreement with experimental data, under mild unfolding conditions, a partial or complete opening of the N-terminal arm is observed, whereas the dislocation of the C-terminus away from the core of the structure occurs only during the low-pH simulations. Furthermore, the picture emerging from this study indicates that the same protein can have different pathways for domain swapping. Indeed, in RNase A the C-terminal swapping requires a substantial unfolding of the monomers, whereas the N-terminal swapping can occur through only partial unfolding. [ABSTRACT FROM AUTHOR]

High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a β-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the β-barrel in azurin is associated with dislocation of its unique α-helix with respect to the protein scaffold. Exposure of the hydrophobic core to solvent precedes complete disruption of secondary and tertiary structure, and modifications in the region around the active site are directly connected with this event. Denaturation of the protein initiates from the sheet coordinating the copper ion, and the other sheet maintains its topology. Results derived from the simulation were compared with experimental data obtained with different techniques, showing excellent agreement and providing a framework to understand the process of disruption and formation of the β-barrel in azurin. [ABSTRACT FROM AUTHOR]

All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described. [Copyright &y& Elsevier]

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

The ability to diagnose and treat prion diseases is limited by our current understanding of the conversion process of the protein from healthy to harmful isoform. Whereas the monomeric, benign species is well characterized, the misfolded conformations responsible for infectivity and neurodegeneration remain elusive. There is mounting evidence that fibrillization intermediates, or protofibrils, but not mature fibrils or plaques, are the pathogenic species in amyloid diseases. Here, we use molecular dynamics to simulate the conversion of the prion protein. Molecular dynamics simulation produces a scrapie prion protein-like conformation enriched in β-structure that is in good agreement with available experimental data. The converted conformation was then used to model a protofibril by means of the docking of hydrophobic patches of the template structure to form hydrogen-bonded sheets spanning adjacent subunits. The resulting protofibril model provides a non-branching aggregate with a 31 axis of symmetry that is in good agreement with a wide variety of experimental data; importantly, it was derived from realistic simulation of the conversion process. [ABSTRACT FROM AUTHOR]

We can track the positions and movements of all the atoms in small proteins as they fold and unfold by combining experimental studies with atomic-resolution molecular dynamics simulations. General principles as to how such complex architectures form so rapidly are now emerging from in-depth studies of a few proteins. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60°C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's T[sub m] of 75°C. The first step in unfolding was expansion of the hydrophobic core. Then, the core was solvated by water and later by urea. The denatured structures in both urea and at high temperature contained residual native helical structure, whereas the β-structure was completely disrupted. The average residence time for urea around hydrophilic groups was six times greater than around hydrophobic residues and in all cases greater than the corresponding water residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure and dynamics, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. In addition, through urea's weakening of water structure, water became free to compete with intraprotein interactions. Urea also interacted directly with polar residues and the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest that urea denatures proteins via both direct and indirect mechanisms. [ABSTRACT FROM AUTHOR]

Proteins appear to fold by diverse pathways, but variations of a simple mechanism – nucleation-condensation – describe the overall features of folding of most domains. In general, secondary structure is inherently unstable and its stability is enhanced by tertiary interactions. Consequently, an extensive interplay of secondary and tertiary interactions determines the transition-state for folding, which is structurally similar to the native state, being formed in a general collapse (condensation) around a diffuse nucleus. As the propensity for stable secondary structure increases, folding becomes more hierarchical and eventually follows a framework mechanism where the transition state is assembled from pre-formed secondary structural elements. [Copyright &y& Elsevier]

Examines the importance and relevance of protein folding in vitro and in silico to in vivo protein folding, misfolding, and disease. Problems in protein folding; Characteristics of transition state; Mapping residual structure in denatured states.

Performs direct comparison of experimental and calculated folding free energies for hydrophobic deletion mutants of chymotrypsin inhibitor 2. Calculation of free energy perturbation using transition and denature-state structures; Conduction of molecular dynamic simulations for the thermal denaturation of enzyme inhibitor.

Focuses on a study that presented multiple native and denaturation simulations of the B and E domains of the three-helix bundle staphylococcal protein A from Staphylococcus aureus. Methodology of the study; Results and discussion; Conclusion.

Investigates the introduction of a de Novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b5. Localized dynamics on the surface of the protein; Periodic formation of a cleft that provides access to the hme through a protected hydrophobic channel; Influences on protein-protein recognition.

Analyzes the folding and unfolding pathway of barnase using a method similar to the classical Bronsted-beta approach. Description of the unfolding of wild-type barnase and the Y17G and 188V mutants; Characterization of the transition state of unfolding; Effect of mutations on the unfolding pathway; Effect of mutations on the transition state of unfolding.

Investigates the structural and dynamical consequences of heme removal by performing molecular dynamics simulations of rat and bovine apocytochrome b5. Availability of a crystal structure for the bovine holoprotein; Correlation of a bovine simulation with rat holocytochrome b5 experimental data; Marked effect on the structure and dynamics of the apo form.

Attempts to study the structural stability and dynamic characteristics of cytochrome b5 which participates in electron -transfer reactions with different proteins by performing molecular dynamics simulation. Methods; Results; Discussion.

Describes a study which used molecular dynamics simulation to model the experimentally observed pH-dependent conformational behavior of amyloid beta(1-28) peptide fragment. Mechanism for the pH-dependent structural rearrangement involving the creation of hydrogen-bonded pair between serine-8 and glutamine-11; Multiequilibrium of folded and unfolded peptide.

Investigates the proposed mechanism for the unfolding of the N-terminal portion of amyloid beta(1-28) peptide at neutral pH. Computational mutagenesis studies; Alteration of the manner with which mutant peptides respond to pH; Importance of serine-6 and glutamine-11 interaction.

Investigates the dynamic behavior of the ribonuclease inhibitor barstar by means of using molecular dynamics (MD) simulations in explicit water. Characteristics of proteins which have been immersed in water; Factors which influence the behavior of ribonuclease inhibitors; Information on MD simulations.