24 results on '"Daggett, Valerie"'
Search Results
2. All-Atom Simulations Of Protein Folding And Unfolding
- Author
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Day, Ryan, primary and Daggett, Valerie, additional
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- 2003
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3. Simulations and computational analyses of prion protein conformations
- Author
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Alonso, Darwin O.V., primary and Daggett, Valerie, additional
- Published
- 2001
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4. Protein Dynamics: A Theoretical Perspective
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Kazmirski, Steven L., primary and Daggett, Valerie, additional
- Published
- 1997
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5. The conformational consequences of mutations to the H1 helix of the prion protein explored by molecular dynamics simulations
- Author
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Kazmirski, Steven L., primary, Alonso, Darwin O.V., additional, Cohen, Fred E., additional, Prusiner, Stanley B., additional, and Daggett, Valerie, additional
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- 1996
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6. A Model for the Molten Globule State of CTF Generated Using Molecular Dynamics
- Author
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Daggett, Valerie, primary
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- 1993
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7. Modulation of post-powerstroke dynamics in myosin II by 2'-deoxy-ADP.
- Author
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Childers MC, Geeves M, Daggett V, and Regnier M
- Subjects
- Adenosine Diphosphate chemistry, Adenosine Diphosphate metabolism, Binding Sites, Deoxyadenine Nucleotides chemistry, Dictyostelium enzymology, Molecular Dynamics Simulation, Myosin Type II chemistry, Pliability, Protein Binding, Protein Conformation drug effects, Protein Domains, Deoxyadenine Nucleotides metabolism, Myosin Type II metabolism
- Abstract
Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin 'sense' changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2'-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle., (Copyright © 2020 Elsevier Inc. All rights reserved.)
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- 2021
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8. Visualizing Protein Folding and Unfolding.
- Author
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Ferina J and Daggett V
- Subjects
- Animals, Humans, Molecular Dynamics Simulation, Protein Conformation, Protein Folding, Protein Unfolding, Proteins chemistry
- 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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9. De Novo Designed α-Sheet Peptides Inhibit Functional Amyloid Formation of Streptococcus mutans Biofilms.
- Author
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Paranjapye N and Daggett V
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- Amyloid ultrastructure, Bacterial Proteins chemistry, Durapatite chemistry, Durapatite metabolism, Humans, Peptides chemical synthesis, Protein Aggregates, Protein Binding, Protein Conformation, beta-Strand, Amyloid chemistry, Amyloid metabolism, Bacterial Proteins metabolism, Biofilms, Peptides chemistry, Peptides metabolism, Streptococcus mutans physiology
- 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
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- 2018
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10. Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms.
- Author
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Bleem A, Christiansen G, Madsen DJ, Maric H, Strømgaard K, Bryers JD, Daggett V, Meyer RL, and Otzen DE
- Subjects
- Amino Acid Sequence, Amyloid chemistry, Amyloid genetics, Amyloid ultrastructure, Amyloidogenic Proteins chemistry, Amyloidogenic Proteins genetics, Amyloidogenic Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Conserved Sequence, Disulfides, Extracellular Matrix, Hydrogen-Ion Concentration, Kinetics, Protein Aggregates, Protein Binding, Protein Multimerization, Amyloid metabolism, Bacterial Proteins metabolism, Biofilms, Protein Engineering, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism
- Abstract
Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections., (Copyright © 2018. Published by Elsevier Ltd.)
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- 2018
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11. Peptides Composed of Alternating L- and D-Amino Acids Inhibit Amyloidogenesis in Three Distinct Amyloid Systems Independent of Sequence.
- Author
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Kellock J, Hopping G, Caughey B, and Daggett V
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- Amino Acid Sequence, Amyloidosis metabolism, Humans, Islet Amyloid Polypeptide metabolism, Molecular Dynamics Simulation, Prealbumin metabolism, Protein Aggregates physiology, Protein Multimerization physiology, Protein Structure, Secondary, Amino Acids metabolism, Amyloid metabolism, Amyloid beta-Peptides metabolism, Amyloidogenic Proteins metabolism
- Abstract
There is now substantial evidence that soluble oligomers are primary toxic agents in amyloid diseases. The development of an antibody recognizing the toxic soluble oligomeric forms of different and unrelated amyloid species suggests a common conformational intermediate during amyloidogenesis. We previously observed a common occurrence of a novel secondary structure element, which we call α-sheet, in molecular dynamics (MD) simulations of various amyloidogenic proteins, and we hypothesized that the toxic conformer is composed of α-sheet structure. As such, α-sheet may represent a conformational signature of the misfolded intermediates of amyloidogenesis and a potential unique binding target for peptide inhibitors. Recently, we reported the design and characterization of a novel hairpin peptide (α1 or AP90) that adopts stable α-sheet structure and inhibits the aggregation of the β-Amyloid Peptide Aβ42 and transthyretin. AP90 is a 23-residue hairpin peptide featuring alternating D- and L-amino acids with favorable conformational propensities for α-sheet formation, and a designed turn. For this study, we reverse engineered AP90 to identify which of its design features is most responsible for conferring α-sheet stability and inhibitory activity. We present experimental characterization (CD and FTIR) of seven peptides designed to accomplish this. In addition, we measured their ability to inhibit aggregation in three unrelated amyloid species: Aβ42, transthyretin, and human islet amylin polypeptide. We found that a hairpin peptide featuring alternating L- and D-amino acids, independent of sequence, is sufficient for conferring α-sheet structure and inhibition of aggregation. Additionally, we show a correlation between α-sheet structural stability and inhibitory activity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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12. Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca(2+)-desensitizing variants.
- Author
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Wang D, McCully ME, Luo Z, McMichael J, Tu AY, Daggett V, and Regnier M
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- Amino Acid Substitution, Animals, Binding Sites, Escherichia coli chemistry, Escherichia coli genetics, Genetic Vectors chemistry, Genetic Vectors genetics, Hydrogen Bonding, Hydrophobic and Hydrophilic Interactions, Molecular Dynamics Simulation, Multiprotein Complexes chemistry, Muscle Contraction, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Isoforms chemistry, Protein Stability, Protein Structure, Secondary, Protein Transport, Rats, Recombinant Proteins chemistry, Recombinant Proteins genetics, Spectrometry, Fluorescence methods, Structure-Activity Relationship, Troponin C genetics, Troponin I chemistry, Troponin I genetics, Calcium chemistry, Protein Interaction Mapping methods, Troponin C chemistry
- Abstract
Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca(2+) binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca(2+) binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca(2+) affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca(2+), but did decrease the interaction in the presence of Ca(2+). In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca(2+). This difference in the absence of Ca(2+) suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca(2+) binding induced by I61Q may result from destabilization of the Ca(2+) binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca(2+) binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca(2+) binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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13. Using simulations to provide the framework for experimental protein folding studies.
- Author
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Rizzuti B and Daggett V
- Subjects
- Models, Molecular, Pressure, Protein Binding, Temperature, Molecular Dynamics Simulation, Protein Folding, Proteins chemistry
- 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 © 2013. Published by Elsevier Inc.)
- Published
- 2013
- Full Text
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14. Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding.
- Author
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van der Kamp MW and Daggett V
- Subjects
- Humans, Models, Molecular, Molecular Dynamics Simulation, Mutant Proteins metabolism, Prions metabolism, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Mutant Proteins chemistry, Mutant Proteins genetics, Mutation, Missense, Prions chemistry, Prions genetics
- 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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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15. Four human thiopurine s-methyltransferase alleles severely affect protein structure and dynamics.
- Author
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Rutherford K and Daggett V
- Subjects
- Alleles, Amino Acid Substitution, Catalytic Domain genetics, Enzyme Stability, Humans, Hydrogen Bonding, Methyltransferases antagonists & inhibitors, Methyltransferases metabolism, Models, Molecular, Pharmacogenetics, Polymorphism, Single Nucleotide, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Thermodynamics, Ubiquitination, Methyltransferases chemistry, Methyltransferases genetics
- 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 alpha3 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 beta-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix alpha8 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.
- Published
- 2008
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16. Direct observation of microscopic reversibility in single-molecule protein folding.
- Author
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Day R and Daggett V
- Subjects
- Computer Simulation, Hydrophobic and Hydrophilic Interactions, Protein Denaturation, Protein Renaturation, Structure-Activity Relationship, Microscopy methods, Models, Molecular, Protein Conformation, Protein Folding
- Abstract
Both folded and unfolded conformations should be observed for a protein at its melting temperature (T(m)), where DeltaG 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 beta-sheet pulls apart and the alpha-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).
- Published
- 2007
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17. Phi-analysis at the experimental limits: mechanism of beta-hairpin formation.
- Author
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Petrovich M, Jonsson AL, Ferguson N, Daggett V, and Fersht AR
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- Amino Acid Sequence, Animals, Guanidine chemistry, Hydrogen-Ion Concentration, Kinetics, Mice, Models, Molecular, Molecular Sequence Data, Mutation genetics, Protein Denaturation, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics, Transcriptional Elongation Factors, Carrier Proteins chemistry
- Abstract
The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.
- Published
- 2006
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18. The folding pathway of spectrin R17 from experiment and simulation: using experimentally validated MD simulations to characterize States hinted at by experiment.
- Author
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Scott KA, Randles LG, Moran SJ, Daggett V, and Clarke J
- Subjects
- Amino Acid Sequence, Animals, Brain Chemistry, Chickens, Models, Molecular, Mutation, Spectrin genetics, Spectrin metabolism, Computer Simulation, Protein Folding, Protein Structure, Tertiary, Spectrin chemistry
- Abstract
We present an experimental and computational analysis of the folding pathway of the 17th domain of chicken brain alpha-spectrin, R17. Wild-type R17 folds in a two-state manner and the chevron plot (plot of the logarithm of the observed rate constant against concentration of urea) shows essentially linear folding and unfolding arms. A number of mutant proteins, however, show a change in slope of the unfolding arm at high concentration of denaturant, hinting at complexity in the folding landscape. Through a combination of mutational studies and high temperature molecular dynamics simulations we show that the folding of R17 can be described by a model with two sequential transition states separated by an intermediate species. The rate limiting transition state for folding in water has been characterized both through experimental Phi-value analysis and by simulation. In contrast, a detailed analysis of the transition state predicted to dominate under highly denaturing conditions is only possible by simulation.
- Published
- 2006
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19. Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding.
- Author
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White GW, Gianni S, Grossmann JG, Jemth P, Fersht AR, and Daggett V
- Subjects
- Hydrogen-Ion Concentration, Kinetics, Magnetic Resonance Spectroscopy, Mutation, Protein Denaturation, Protein Structure, Tertiary, Proto-Oncogene Proteins c-myb chemistry, Proto-Oncogene Proteins c-myb metabolism, Temperature, Time Factors, Urea, Computer Simulation, Protein Folding
- Abstract
There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.
- Published
- 2005
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20. The structure of the major transition state for folding of an FF domain from experiment and simulation.
- Author
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Jemth P, Day R, Gianni S, Khan F, Allen M, Daggett V, and Fersht AR
- Subjects
- Carrier Proteins chemistry, Carrier Proteins genetics, Carrier Proteins metabolism, Glycine genetics, Glycine metabolism, Humans, Kinetics, Models, Molecular, Mutation genetics, Protein Denaturation, Protein Engineering, Protein Renaturation, Protein Structure, Secondary, Thermodynamics, Computer Simulation, Protein Folding, Protein Structure, Tertiary
- Abstract
We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Phi-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (PhiF=0.9-1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (PhiF=0-0.2). The hydrophobic core and other parts of the protein displayed intermediate Phi-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Phi-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation-condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Phi-values.
- Published
- 2005
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21. Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation.
- Author
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Ferguson N, Day R, Johnson CM, Allen MD, Daggett V, and Fersht AR
- Subjects
- Bacterial Proteins genetics, Calorimetry, Differential Scanning, Circular Dichroism, Computer Simulation, Geobacillus stearothermophilus chemistry, Geobacillus stearothermophilus genetics, Hydrogen Bonding, Kinetics, Models, Molecular, Mutation genetics, Nuclear Magnetic Resonance, Biomolecular, Protein Denaturation, Protein Structure, Tertiary, Thermodynamics, Time Factors, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Hot Temperature, Protein Folding
- Abstract
We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.
- Published
- 2005
- Full Text
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22. Diffusing and colliding: the atomic level folding/unfolding pathway of a small helical protein.
- Author
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DeMarco ML, Alonso DO, and Daggett V
- Subjects
- Protein Folding, Proteins metabolism, Proteins chemistry
- Abstract
Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion-collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 micros of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD., (Copyright 2004 Elsevier Ltd.)
- Published
- 2004
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23. Increasing temperature accelerates protein unfolding without changing the pathway of unfolding.
- Author
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Day R, Bennion BJ, Ham S, and Daggett V
- Subjects
- Binding Sites, Kinetics, Models, Molecular, Movement, Plant Proteins, Protein Denaturation, Protein Structure, Secondary, Software, Solvents chemistry, Solvents metabolism, Thermodynamics, Water chemistry, Water metabolism, Computer Simulation, Peptides chemistry, Peptides metabolism, Protein Folding, Temperature
- Abstract
We have traditionally relied on extremely elevated temperatures (498K, 225 degrees C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298K to 498K. The simulation lengths vary from 94ns to 20ns, for a total simulation time of 344ns, or 0.34 micros. At 298K, the protein is very stable over the full 50ns simulation. At 348K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25ns, explores partially unfolded conformations for 20ns, and then refolds over the last 35ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.
- Published
- 2002
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24. Probing the energy landscape of protein folding/unfolding transition states.
- Author
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De Jong D, Riley R, Alonso DO, and Daggett V
- Subjects
- Animals, Computer Simulation, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Protein Conformation, Protein Denaturation, Protein Renaturation, Swine, Temperature, Thermodynamics, Time Factors, Tryptophan chemistry, Water chemistry, Protein Folding, Protein Precursors chemistry, Protein Precursors metabolism
- Abstract
Previous molecular dynamics (MD) simulations of the thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided atomic-resolution models of the transition state ensemble that is well supported by experimental studies. Here, we use simulations to further investigate the energy landscape around the transition state region. Nine structures within approximately 35 ps and 3 A C(alpha) RMSD of the transition state ensemble identified in a previous 498 K thermal denaturation simulation were quenched under the quasi-native conditions of 335 K and neutral pH. All of the structures underwent hydrophobically driven collapse in response to the drop in temperature. Structures less denatured than the transition state became structurally more native-like, while structures that were more denatured than the transition state tended to show additional loss of native structure. The structures in the immediate region of the transition state fluctuated between becoming more and less native-like. All of the starting structures had the same native-like topology and were quite similar (within 3.5 A C(alpha) RMSD). That the structures all shared native-like topology, yet diverged into either more or less native-like structures depending on which side of the transition state they occupied on the unfolding trajectory, indicates that topology alone does not dictate protein folding. Instead, our results suggest that a detailed interplay of packing interactions and interactions with water determine whether a partially denatured protein will become more native-like under refolding conditions.
- Published
- 2002
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