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All currently known structures of proteins together define ‘protein fold space’. To increase the general understanding of protein dynamics and protein folding, we selected a set of 807 proteins and protein domains that represent 95% of the currently known autonomous folded domains present in globular proteins. Native state and unfolding simulations of these representatives are now complete and accessible via a novel database containing over 11 000 simulations. Because protein folding is a microscopically reversible process, these simulations effectively sample protein folding across all of protein fold space. Here, we give an overview of how the representative proteins were selected and how the simulations were performed and validated. We then provide examples of different types of analyses that can be performed across our large set of simulations, made possible by the database approach. We further show how the unfolding simulations can be used to compare unfolding of structural elements in isolation and in different structural contexts, using as an example a short, triple stranded β-sheet that forms the WW domain and is present in several larger unrelated proteins.

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long‐range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures. [ABSTRACT FROM AUTHOR]

Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. Here we used time‐resolved small‐angle X‐ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first‐order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2‐deoxy‐ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X‐ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin‐binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions. Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. [ABSTRACT FROM AUTHOR]

The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation‐associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA‐binding domain. Here, we report molecular dynamics simulations of wild‐type (WT) p53 and 20 different mutants, including a stabilized pseudo‐WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural‐disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural‐disruption motifs rather than a multitude of effects specific to each mutant. In addition, α‐sheet secondary structure formed in almost all of the proteins. α‐Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re‐consideration of cancer as an amyloid disease. [ABSTRACT FROM AUTHOR]

The Dynameomics project contains native state and unfolding simulations of 807 protein domains, where each domain is representative of a different metafold; these metafolds encompass ~97% of protein fold space. There is a long-standing question in structural biology as to whether proteins in the same fold family share the same folding/unfolding characteristics. Using molecular dynamics simulations from the Dynameomics project, we conducted a detailed study of protein unfolding/folding pathways for 5 protein domains from the immunoglobulin (Ig)-like β-sandwich metafold (the highest ranked metafold in our database). The domains have sequence similarities ranging from 4 to 15% and are all from different SCOP superfamilies, yet they share the same overall Ig-like topology. Despite having very different amino acid sequences, the dominant unfolding pathway is very similar for the 5 proteins, and the secondary structures that are peripheral to the aligned, shared core domain add variability to the unfolding pathway. Aligned residues in the core domain display consensus structure in the transition state primarily through conservation of hydrophobic positions. Commonalities in the obligate folding nucleus indicate that insights into the major events in the folding/unfolding of other domains from this metafold may be obtainable from unfolding simulations of a few representative proteins. [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]

Computational resources have contributed to the design and engineering of novel proteins by integrating genomic, structural and dynamic aspects of proteins. Non-canonical amino acids, such as d -amino acids, expand the available sequence space for designing and engineering proteins; however, the rotamer libraries for d -amino acids are usually constructed as the mirror images of l -amino acid rotamer libraries, an assumption that has not been tested. To this end, we have performed molecular dynamics (MD) simulations of model host–guest peptide systems containing d -amino acids. Our simulations systematically address the applicability of the mirror image convention as well as the effects of neighboring residue chirality. Rotamer libraries derived from these systems provide realistic rotamer distributions suitable for use in both rational and computational design workflows. Our simulations also address the impact of chirality on the intrinsic conformational preferences of amino acids, providing fundamental insights into the relationship between chirality and biomolecular dynamics. While d -amino acids are rare in naturally occurring proteins, they are used in designed proteins to stabilize a desired conformation, increase bioavailability or confer favorable biochemical and physical attributes. Here, we present d -amino acid rotamer libraries derived from MD simulations of alanine-based host–guest pentapeptides and show how certain residues can deviate from mirror image symmetry. Our simulations directly model d -amino acids as guest residues within the chiral l -Ala and d -Ala pentapeptide series to explicitly incorporate any contributions resulting from the chiralities of neighboring residues. [ABSTRACT FROM AUTHOR]

Prion diseases are associated with the misfolding of the prion protein (PrP) from its normal cellular form (Pr PC) to its infectious scrapie form (Pr PSc). Post-translational modifications in PrP in vivo can play an important role in modulating the process of misfolding. To gain more insight into the effects of post-translational modifications in PrP structure and dynamics and to test the hypothesis that such modifications can interact with the protein, we have performed molecular dynamics simulations of diglycosylated human Pr PC bound to a lipid bilayer via a glycophosphatidylinositol anchor. Multiple simulations were performed at three different pH ranges to explore pH effects on structure and dynamics. In contrast to simulations of protein-only Pr PC, no large effects were observed upon lowering the pH of the system. The protein tilted toward the membrane surface in all of the simulations and the putative Pr PSc oligomerization sites became inaccessible, thereby offering a possible protective mechanism against Pr PSc-induced misfolding of PrPC. [ABSTRACT FROM AUTHOR]

d-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of l- and d-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host–guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the d-amino acids, including d-allo-isoleucine and d-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate l-amino acids verifies that the intrinsic backbone conformational propensities of the d-amino acids are the inverse of their cognate l-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties. [ABSTRACT FROM PUBLISHER]

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]

The need for data-centric scientific tools is growing; domains such as biology, chemistry, and physics are increasingly adopting computational approaches. So, scientists must deal with the challenges of big data. To address these challenges, researchers built a visual-analytics platform named DIVE (Data Intensive Visualization Engine). DIVE is a data-agnostic, ontologically expressive software framework that can stream large datasets at interactive speeds. In particular, DIVE makes novel contributions to structured-data-model manipulation and high-throughput streaming of large, structured datasets. [ABSTRACT FROM AUTHOR]

There are over 100 mutations in Cu/Zn superoxide dismutase (SOD1) that result in a subset of familial amyotrophic lateral sclerosis (fALS) cases. The hypothesis that dissociation of the dimer, misfolding of the monomer and subsequent aggregation of mutant SOD1 leads to fALS has been gaining support as an explanation for how these disparate missense mutations cause the same disease. These forms are only responsible for a fraction of the ALS cases; however, the rest are sporadic. Starting with a folded apo monomer, the species considered most likely to be involved in misfolding, we used high-temperature all-atom molecular dynamics simulations to explore the events of the wild-type protein unfolding through the denatured state. All simulations showed early loss of structure along the β5–β6 edge of the β-sandwich, supporting earlier findings of instability in this region. Transition state structures identified from the simulations are in good agreement with experiment, providing detailed, validated molecular models for this elusive state. Furthermore, we compare the process of thermal unfolding investigated here to that of the lethal A4V mutant-induced unfolding at physiological temperature and find that the pathways are very similar. [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]

A thermostabilized variant (UVF) of the engrailed homeodomain (EnHD) was previously engineered by Mayo and co-workers. The melting temperature of the non-natural, designed protein is 50°C higher than the natural wild-type protein (>99 vs. 52°C), and the two proteins share 22% sequence identity. We have performed extensive (1 μs) all-atom, explicit solvent molecular dynamics simulations of the wild-type and engineered proteins to investigate their structural and dynamic properties at room temperature and at 100°C. Our simulations are in good agreement with nuclear magnetic resonance data available for the two proteins [nuclear Overhauser effect crosspeaks (NOEs), J-coupling constants and order parameters for EnHD; and NOEs for UVF], showing that we reproduce the backbone dynamics and side chain packing in the native state of both proteins. UVF was more dynamic at room temperature than EnHD, with respect to both its backbone and side chain motion. When the temperature was raised, the thermostable protein maintained this mobility while retaining its native conformation. EnHD, on the other hand, was unable to maintain its more rigid native structure at higher temperature and began to unfold. Heightened protein dynamics leading to promiscuous and dynamically interchangeable amino acid contacts makes UVF more tolerant to increasing temperature, providing a molecular explanation for heightened thermostability of this protein. [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]

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 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]

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]

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.

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]

To better characterize the transition state for folding/unfolding and its sensitivity to environmental changes, we have run multiple molecular dynamics simulations of chymotrypsin inhibitor 2 (CI2) under varying solvent conditions and temperature. The transition state structures agree well with experiment, and are similar under all of the conditions investigated here. Increasing the temperature leads to some movement in the position of the transition state along several reaction coordinates, as measured by changes in properties of the transition state structures. These structural changes are in the direction of a more native-like transition state as denaturation conditions become more severe, as expected for a Hammond effect. These structural changes are not, however, reflected in the global structure as measured by the total number of contacts or the average S-values. These results suggest that the small changes in average Φ-values with temperature seen by experiment may be due to an increase in the sensitivity of the transition state to mutation rather than a change in the average structure of the transition state. A simple analysis of the rates of unfolding indicates that the free energy barrier to unfolding decreases with increasing temperature, but even in our very high temperature simulations there is a small free energy barrier. [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]

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]

Here, we describe the folding/unfolding kinetics of α[SUB3]D, a small designed three-helix bundle. Both IR temperature jump and ultra-fast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 ± 1.2 μs (at ≈ 50°C), indicating that a protein can fold on the 1- to 5-μs time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably ≥ 1 (μs)1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. α[SUB3]D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways. [ABSTRACT FROM AUTHOR]