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Dirk Schneider - One of the best experts on this subject based on the ideXlab platform.

  • Transmembrane Helix-Helix interactions are modulated by the sequence context and by lipid bilayer properties.
    Biochimica et biophysica acta, 2011
    Co-Authors: Florian Cymer, Anbazhagan Veerappan, Dirk Schneider
    Abstract:

    Abstract Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM HelixHelix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane HelixHelix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane HelixHelix interaction. Structures of 11 transmembrane Helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes.

  • A mutational study of transmembrane HelixHelix interactions
    Biochimie, 2007
    Co-Authors: Alexander Prodöhl, Mathias Weber, Carolin Dreher, Dirk Schneider
    Abstract:

    Abstract Diverse methods have been developed and applied in the recent years to study interaction of transmembrane α-helices and often interaction of single transmembrane helices is followed on SDS-gels. Here we compare two measurements of the stability of a transmembrane HelixHelix interaction, and the stability of the PsbF transmembrane Helix dimer was determined in a biological membrane as well as in SDS. The observations described in this study demonstrate that the environment, in which a transmembrane Helix interaction is studied, can be very critical and detergent properties can significantly influence transmembrane Helix interactions, especially, when the transmembrane domain contains strongly polar residues.

Pramodh Vallurupalli - One of the best experts on this subject based on the ideXlab platform.

  • atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in t4 lysozyme
    PLOS Computational Biology, 2018
    Co-Authors: Jagannath Mondal, Navjeet Ahalawat, Subhendu Pandit, Lewis E Kay, Pramodh Vallurupalli
    Abstract:

    Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (T4L L99A) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound T4L L99A are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by T4L L99A at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of T4L L99A. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 μs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (Helix4/Helix6, Helix7/Helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in wild type T4L. Taken together and via integrating with results from experiments, these simulations provide unprecedented mechanistic insights into the complete ligand recognition process in a buried cavity. By illustrating the power of subtle Helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition in a solvent-inaccessible cavity, contrary to the common perception of a single dominant pathway for ligand binding.

  • atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in t4 lysozyme
    bioRxiv, 2018
    Co-Authors: Jagannath Mondal, Navjeet Ahalawat, Subhendu Pandit, Lewis E Kay, Pramodh Vallurupalli
    Abstract:

    Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (L99A T4L) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound L99A T4L are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by L99A T4L at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of L99A T4L. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 μs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (Helix4/Helix6, Helix7/Helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in otherwise ligand-inactive wild type T4L. Taken together, by integrating experiments, these simulations provide unprecedented mechanistic insights into complete ligand recognition process in a buried cavity. By illustrating the power of subtle Helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition mechanism in a solvent-inaccessible cavity, contrary to common perception of single dominant pathway for ligand binding.

Jinhyuk Lee - One of the best experts on this subject based on the ideXlab platform.

  • Role of Hydrogen Bonding and Helix−Lipid Interactions in Transmembrane Helix Association
    Journal of the American Chemical Society, 2008
    Co-Authors: Jinhyuk Lee
    Abstract:

    To explore the role of hydrogen bonding and Helix−lipid interactions in transmembrane Helix association, we have calculated the potential of mean force (PMF) as a function of HelixHelix distance between two pVNVV peptides, a transmembrane model peptide based on the GCN4 leucine-zipper, in a dimyristoylphosphatidylcholine (DMPC) membrane. The peptide name pVNVV represents the interfacial residues in the heptad repeat of the dimer. The free energy decomposition reveals that the total PMF consists of two competing contributions from HelixHelix and Helix−lipid interactions. The direct, favorable HelixHelix interactions arise from the specific contribution from the Helix-facing residues and the generic contribution from the lipid-facing residues. The Asn residues in the middle of the helices show the most significant per-residue contribution to the PMF with various hydrogen bonding patterns as a function of HelixHelix distance. Release of lipid molecules between the helices into bulk lipid upon Helix assoc...

  • implementation and application of Helix Helix distance and crossing angle restraint potentials
    Journal of Computational Chemistry, 2007
    Co-Authors: Jinhyuk Lee
    Abstract:

    Based on the definition of HelixHelix distance and crossing angle introduced by Chothia et al. (J Mol Biol 1981, 145, 215), we have developed the restraint potentials by which the distance and crossing angle of two selected helices can be maintained around target values during molecular dynamics simulations. A series of assessments show that calculated restraint forces are numerically accurate. Since the restraint forces are only exerted on atoms which define the helical principal axes, each Helix can rotate along its helical axis, depending on the HelixHelix intermolecular interactions. Such a restraint potential enables us to characterize the HelixHelix interactions at atomic details by sampling their conformational space around specific distance and crossing angle with (restraint) force-dependent fluctuations. Its efficacy is illustrated by calculating the potential of mean force as a function of HelixHelix distance between two transmembrane helical peptides in an implicit membrane model. © 2006 Wiley Periodicals, Inc. J Comput Chem 28: 669–680, 2007

  • Implementation and application of HelixHelix distance and crossing angle restraint potentials
    Journal of computational chemistry, 2006
    Co-Authors: Jinhyuk Lee
    Abstract:

    Based on the definition of HelixHelix distance and crossing angle introduced by Chothia et al. (J Mol Biol 1981, 145, 215), we have developed the restraint potentials by which the distance and crossing angle of two selected helices can be maintained around target values during molecular dynamics simulations. A series of assessments show that calculated restraint forces are numerically accurate. Since the restraint forces are only exerted on atoms which define the helical principal axes, each Helix can rotate along its helical axis, depending on the HelixHelix intermolecular interactions. Such a restraint potential enables us to characterize the HelixHelix interactions at atomic details by sampling their conformational space around specific distance and crossing angle with (restraint) force-dependent fluctuations. Its efficacy is illustrated by calculating the potential of mean force as a function of HelixHelix distance between two transmembrane helical peptides in an implicit membrane model. © 2006 Wiley Periodicals, Inc. J Comput Chem 28: 669–680, 2007

Jagannath Mondal - One of the best experts on this subject based on the ideXlab platform.

  • atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in t4 lysozyme
    PLOS Computational Biology, 2018
    Co-Authors: Jagannath Mondal, Navjeet Ahalawat, Subhendu Pandit, Lewis E Kay, Pramodh Vallurupalli
    Abstract:

    Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (T4L L99A) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound T4L L99A are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by T4L L99A at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of T4L L99A. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 μs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (Helix4/Helix6, Helix7/Helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in wild type T4L. Taken together and via integrating with results from experiments, these simulations provide unprecedented mechanistic insights into the complete ligand recognition process in a buried cavity. By illustrating the power of subtle Helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition in a solvent-inaccessible cavity, contrary to the common perception of a single dominant pathway for ligand binding.

  • atomic resolution mechanism of ligand binding to a solvent inaccessible cavity in t4 lysozyme
    bioRxiv, 2018
    Co-Authors: Jagannath Mondal, Navjeet Ahalawat, Subhendu Pandit, Lewis E Kay, Pramodh Vallurupalli
    Abstract:

    Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent. Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (L99A T4L) that rapidly binds benzene (~106 M-1s-1). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound L99A T4L are available, the kinetic pathways by which benzene reaches its solvent-inaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by L99A T4L at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of L99A T4L. The simulated and high-resolution X-ray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 μs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (Helix4/Helix6, Helix7/Helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in otherwise ligand-inactive wild type T4L. Taken together, by integrating experiments, these simulations provide unprecedented mechanistic insights into complete ligand recognition process in a buried cavity. By illustrating the power of subtle Helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition mechanism in a solvent-inaccessible cavity, contrary to common perception of single dominant pathway for ligand binding.

Alan R Fersht - One of the best experts on this subject based on the ideXlab platform.

  • kinetics of chain motions within a protein folding intermediate
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Hannes Neuweiler, Wiktor Banachewicz, Alan R Fersht
    Abstract:

    Small proteins can fold remarkably rapidly, even in μs. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the Helix2-turn-Helix3 motif of the 3-Helix bundle forms in approximately 2 μs, but the final docking of preformed Helix1 in I requires approximately 20 μs. Simulation and structural data suggest that nonnative interactions may slow down Helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in Helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-μs time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-μs and 200-ns phase for labels in the Helix2-turn-Helix3 motif of I, because the native state is not significantly populated. But for labels in Helix1 we observed an additional approximately 10-μs phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.

  • Helix Stability in Barstar Peptides
    FEBS Journal, 1997
    Co-Authors: Andrés Santos Soler-gonzález, Alan R Fersht
    Abstract:

    The complex between the ribonuclease barnase and barstar, its intracellular inhibitor, is a very good model for studying protein folding and molecular recognition. We have studied the stability of different peptides that cover the barstar α-Helix2 involved in the binding to barnase. A linear correlation between the helical amphipathy of these peptides and their inhibitory ability was obtained: the more helically amphipathic, the more the affinity for barnase. We estimated the amount of Helix of these peptides in water and in trifluoroethanol by circular dichroism. There is a moderate correlation between the helical amphipathy and the helical content in water, in agreement with previous results that have shown the importance of the hydrophobicity periodicity in the design of peptides. The helical content in trifluoroethanol is related to helical propensity and helical amphipathy, suggesting that the local sequence determines these maximum helicities. The predicted helicity of these peptides, obtained using the algorithm AGADIR [Mufioz, V. & Serrano, L. (1994) Nat. Struct. Biol. 1, 399–409], appears to correlate with their ability to inhibit the activity of barnase in water. The correlation of inhibition constants, helical content in water, and maximum content of Helix in trifluoroethanol with helical amphipathy supports the very important role of hydrophobicity pattern in peptide stability.

  • the folding of an enzyme v h 2h exchange nuclear magnetic resonance studies on the folding pathway of barnase complementarity to and agreement with protein engineering studies
    Journal of Molecular Biology, 1992
    Co-Authors: Andreas T Matouschek, Luis Serrano, Elizabeth M Meiering, Mark Bycroft, Alan R Fersht
    Abstract:

    Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of Helix2; (2) early formation of Helix3; (3) early formation of several β-turns (46–49, 101–104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) Helix1 is complete from residues 10 to 18; (2) the interactions between all β-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and solH2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.