Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

PubMed

Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

2017-09-09

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Artificial enzymes based on supramolecular scaffolds.

PubMed

Dong, Zeyuan; Luo, Quan; Liu, Junqiu

2012-12-07

Enzymes are nanometer-sized molecules with three-dimensional structures created by the folding and self-assembly of polymeric chain-like components through supramolecular interactions. They are capable of performing catalytic functions usually accompanied by a variety of conformational states. The conformational diversities and complexities of natural enzymes exerted in catalysis seriously restrict the detailed understanding of enzymatic mechanisms in molecular terms. A supramolecular viewpoint is undoubtedly helpful in understanding the principle of enzyme catalysis. The emergence of supramolecular artificial enzymes therefore provides an alternative way to approach the structural complexity and thus to unravel the mystery of enzyme catalysis. This critical review covers the recent development of artificial enzymes designed based on supramolecular scaffolds ranging from the synthetic macrocycles to self-assembled nanometer-sized objects. Such findings are anticipated to facilitate the design of supramolecular artificial enzymes as well as their potential uses in important fields, such as manufacturing and food industries, environmental biosensors, pharmaceutics and so on.

Locking the Active Conformation of c-Src Kinase through the Phosphorylation of the Activation Loop

PubMed Central

Meng, Yilin; Roux, Benoît

2013-01-01

Molecular dynamics umbrella sampling simulations are used to compare the relative stability of the active conformation of the catalytic domain of c-Src kinase while the tyrosine 416 in the activation loop (A-loop) is either unphosphorylated or phosphorylated. When the A-loop is unphosphorylated, there is considerable flexiblity of the kinase. While the active conformation of the kinase is not forbidden and can be visited transiently, it is not the predominant state. This is consistent with the view that c-Src displays some catalytic activity even when the A-loop is unphosphorylated. In contrast, phosphorylation of the A-loop contributes to stabilize several structural features that are critical for catalysis, such as the hydrophobic regulatory spine, the HRD motif, and the electrostatic switch. In summary, the free energy landscape calculations demonstrate that phosphorylation of tyrosine 416 in the A-loop essentially “locks” the kinase into its catalytically competent conformation. PMID:24103328

Protein Conformational Populations and Functionally Relevant Sub-states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K; Burger, Virginia; Savol, Andrej

2013-01-01

Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape. Emerging evidence indicates that protein landscapes contain conformational substates with dynamic and structural features that support the designated function of themore » protein. Nuclear magnetic resonance (NMR) experiments provide information about conformational ensembles of proteins. X-ray crystallography allows researchers to identify the most populated states along the landscape, and computational simulations give atom-level information about the conformational substates of different proteins. This ability to characterize and obtain quantitative information about the conformational substates and the populations of proteins within them is allowing researchers to better understand the relationship between protein structure and dynamics and the mechanisms of protein function. In this Account, we discuss recent developments and challenges in the characterization of functionally relevant conformational populations and substates of proteins. In some enzymes, the sampling of functionally relevant conformational substates is connected to promoting the overall mechanism of catalysis. For example, the conformational landscape of the enzyme dihydrofolate reductase has multiple substates, which facilitate the binding and the release of the cofactor and substrate and catalyze the hydride transfer. For the enzyme cyclophilin A, computational simulations reveal that the long time scale conformational fluctuations enable the enzyme to access conformational substates that allow it to attain the transition state, therefore promoting the reaction mechanism. In the long term, this emerging view of proteins with conformational substates has broad implications for improving our understanding of enzymes, enzyme engineering, and better drug design. Researchers have already used photoactivation to modulate protein conformations as a strategy to develop a hypercatalytic enzyme. In addition, the alteration of the conformational substates through binding of ligands at locations other than the active site provides the basis for the design of new medicines through allosteric modulation.« less

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

PubMed

Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

2016-04-01

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calcium-independent metal-ion catalytic mechanism of anthrax edema factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Yuequan; Zhukovskaya, Natalia L.; Guo, Qing

2009-11-18

Edema factor (EF), a key anthrax exotoxin, has an anthrax protective antigen-binding domain (PABD) and a calmodulin (CaM)-activated adenylyl cyclase domain. Here, we report the crystal structures of CaM-bound EF, revealing the architecture of EF PABD. CaM has N- and C-terminal domains and each domain can bind two calcium ions. Calcium binding induces the conformational change of CaM from closed to open. Structures of the EF-CaM complex show how EF locks the N-terminal domain of CaM into a closed conformation regardless of its calcium-loading state. This represents a mechanism of how CaM effector alters the calcium affinity of CaM andmore » uncouples the conformational change of CaM from calcium loading. Furthermore, structures of EF-CaM complexed with nucleotides show that EF uses two-metal-ion catalysis, a prevalent mechanism in DNA and RNA polymerases. A histidine (H351) further facilitates the catalysis of EF by activating a water to deprotonate 3'OH of ATP. Mammalian adenylyl cyclases share no structural similarity with EF and they also use two-metal-ion catalysis, suggesting the catalytic mechanism-driven convergent evolution of two structurally diverse adenylyl cyclases.« less

Homochiral polymerization-driven selective growth of graphene nanoribbons

NASA Astrophysics Data System (ADS)

Sakaguchi, Hiroshi; Song, Shaotang; Kojima, Takahiro; Nakae, Takahiro

2017-01-01

The surface-assisted bottom-up fabrication of graphene nanoribbons (GNRs), which consists of the radical polymerization of precursors followed by dehydrogenation, has attracted attention because of the method's ability to control the edges and widths of the resulting ribbon. Although these reactions on a metal surface are believed to be catalytic, the mechanism has remained unknown. Here, we demonstrate 'conformation-controlled surface catalysis': the two-zone chemical vapour deposition of a 'Z-bar-linkage' precursor, which represents two terphenyl units linked in a 'Z' shape, results in the efficient formation of acene-type GNRs with a width of 1.45 nm through optimized cascade reactions. These precursors exhibit flexibility that allows them to adopt chiral conformations with height asymmetry on a Au(111) surface, which enables the production of self-assembled homochiral polymers in a chain with a planar conformation, followed by dehydrogenation via a conformation-controlled mechanism. This is conceptually analogous to enzymatic catalysis and will be useful for the fabrication of new nanocarbon materials.

Dynamically achieved active site precision in enzyme catalysis.

PubMed

Klinman, Judith P

2015-02-17

CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

The Structure of a High Fidelity DNA Polymerase Bound to a Mismatched Nucleotide Reveals an “Ajar” Intermediate Conformation in the Nucleotide Selection Mechanism*

PubMed Central

Wu, Eugene Y.; Beese, Lorena S.

2011-01-01

To achieve accurate DNA synthesis, DNA polymerases must rapidly sample and discriminate against incorrect nucleotides. Here we report the crystal structure of a high fidelity DNA polymerase I bound to DNA primer-template caught in the act of binding a mismatched (dG:dTTP) nucleoside triphosphate. The polymerase adopts a conformation in between the previously established “open” and “closed” states. In this “ajar” conformation, the template base has moved into the insertion site but misaligns an incorrect nucleotide relative to the primer terminus. The displacement of a conserved active site tyrosine in the insertion site by the template base is accommodated by a distinctive kink in the polymerase O helix, resulting in a partially open ternary complex. We suggest that the ajar conformation allows the template to probe incoming nucleotides for complementarity before closure of the enzyme around the substrate. Based on solution fluorescence, kinetics, and crystallographic analyses of wild-type and mutant polymerases reported here, we present a three-state reaction pathway in which nucleotides either pass through this intermediate conformation to the closed conformation and catalysis or are misaligned within the intermediate, leading to destabilization of the closed conformation. PMID:21454515

Dynamically Achieved Active Site Precision in Enzyme Catalysis

PubMed Central

2015-01-01

Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

PubMed Central

Sripathi, Kamali N.; Tay, Wendy W.; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G.

2014-01-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2′-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. PMID:24854621

X-ray Crystal Structure of Aristolochene Synthase from Aspergillus terreus and Evolution of Templates for the Cyclization of Farnesyl Diphosphate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishova,E.; Di Costanzo, L.; Cane, D.

2007-01-01

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 {angstrom} resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the {alpha}-helical class I terpene synthase fold with the active site in the 'open', solvent-exposed conformation. Intriguingly, the 2.15 {angstrom} resolution crystal structure of the complex with Mg{sup 2+}{sub 3}-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the 'closed' conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition frommore » the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved {alpha}-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.« less

Protein conformational disorder and enzyme catalysis.

PubMed

Schulenburg, Cindy; Hilvert, Donald

2013-01-01

Though lacking a well-defined three-dimensional structure, intrinsically unstructured proteins are ubiquitous in nature. These molecules play crucial roles in many cellular processes, especially signaling and regulation. Surprisingly, even enzyme catalysis can tolerate substantial disorder. This observation contravenes conventional wisdom but is relevant to an understanding of how protein dynamics modulates enzyme function. This chapter reviews properties and characteristics of disordered proteins, emphasizing examples of enzymes that lack defined structures, and considers implications of structural disorder for catalytic efficiency and evolution.

Mechanical catalysis on the centimetre scale

PubMed Central

Miyashita, Shuhei; Audretsch, Christof; Nagy, Zoltán; Füchslin, Rudolf M.; Pfeifer, Rolf

2015-01-01

Enzymes play important roles in catalysing biochemical transaction paths, acting as logical machines through the morphology of the processes. A key challenge in elucidating the nature of these systems, and for engineering manufacturing methods inspired by biochemical reactions, is to attain a comprehensive understanding of the stereochemical ground rules of enzymatic reactions. Here, we present a model of catalysis that can be performed magnetically by centimetre-sized passive floating units. The designed system, which is equipped with permanent magnets only, passively obeys the local causalities imposed by magnetic interactions, albeit it shows a spatial behaviour and an energy profile analogous to those of biochemical enzymes. In this process, the enzyme units trigger physical conformation changes of the target by levelling out the magnetic potential barrier (activation potential) to a funnel type and, thus, induce cascading conformation changes of the targeted substrate units reacting in parallel. The inhibitor units, conversely, suppress such changes by increasing the potential. Because the model is purely mechanical and established on a physics basis in the absence of turbulence, each performance can be explained by the morphology of the unit, extending the definition of catalysis to systems of alternative scales. PMID:25652461

Mechanical catalysis on the centimetre scale.

PubMed

Miyashita, Shuhei; Audretsch, Christof; Nagy, Zoltán; Füchslin, Rudolf M; Pfeifer, Rolf

2015-03-06

Enzymes play important roles in catalysing biochemical transaction paths, acting as logical machines through the morphology of the processes. A key challenge in elucidating the nature of these systems, and for engineering manufacturing methods inspired by biochemical reactions, is to attain a comprehensive understanding of the stereochemical ground rules of enzymatic reactions. Here, we present a model of catalysis that can be performed magnetically by centimetre-sized passive floating units. The designed system, which is equipped with permanent magnets only, passively obeys the local causalities imposed by magnetic interactions, albeit it shows a spatial behaviour and an energy profile analogous to those of biochemical enzymes. In this process, the enzyme units trigger physical conformation changes of the target by levelling out the magnetic potential barrier (activation potential) to a funnel type and, thus, induce cascading conformation changes of the targeted substrate units reacting in parallel. The inhibitor units, conversely, suppress such changes by increasing the potential. Because the model is purely mechanical and established on a physics basis in the absence of turbulence, each performance can be explained by the morphology of the unit, extending the definition of catalysis to systems of alternative scales.

Accessing protein conformational ensembles using room-temperature X-ray crystallography

PubMed Central

Fraser, James S.; van den Bedem, Henry; Samelson, Avi J.; Lang, P. Therese; Holton, James M.; Echols, Nathaniel; Alber, Tom

2011-01-01

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation. PMID:21918110

Importance of Loop L1 Dynamics for Substrate Capture and Catalysis in Pseudomonas aeruginosa d-Arginine Dehydrogenase.

PubMed

Ouedraogo, Daniel; Souffrant, Michael; Vasquez, Sheena; Hamelberg, Donald; Gadda, Giovanni

2017-05-16

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k cat /K m values with d-arginine were ∼13-fold lower than in wild-type PaDADH. Moreover, the pH profiles for the k cat value with d-arginine showed a hollow, consistent with restricted proton movements in catalysis, and no saturation was achieved with the alternate substrate d-leucine in the reductive half-reaction of the variant enzymes. Taken together, the computational and experimental data are consistent with the dynamics of loop L1 being important for substrate capture and catalysis in PaDADH.

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases.

PubMed

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert; Rovira, Carme

2015-02-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer-Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2 S O → [ 2,5 B] ǂ → 5 S 1 and 1 S 3 → [ 4 H 3 ] ǂ → 4 C 1 , which clearly appear in low energy regions of the FEL. Consistently, 2 S O and 1 S 3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the O E → [ O S 2 ] ǂ → B 2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed O E conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases.

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape.

PubMed

Sripathi, Kamali N; Tay, Wendy W; Banáš, Pavel; Otyepka, Michal; Šponer, Jiří; Walter, Nils G

2014-07-01

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape. © 2014 Sripathi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc02240h Click here for additional data file.

PubMed Central

Iglesias-Fernández, Javier; Raich, Lluís; Ardèvol, Albert

2015-01-01

Unraveling the conformational catalytic itinerary of glycoside hydrolases (GHs) is a growing topic of interest in glycobiology, with major impact in the design of GH inhibitors. β-xylanases are responsible for the hydrolysis of glycosidic bonds in β-xylans, a group of hemicelluloses of high biotechnological interest that are found in plant cell walls. The precise conformations followed by the substrate during catalysis in β-xylanases have not been unambiguously resolved, with three different pathways being proposed from structural analyses. In this work, we compute the conformational free energy landscape (FEL) of β-xylose to predict the most likely catalytic itineraries followed by β-xylanases. The calculations are performed by means of ab initio metadynamics, using the Cremer–Pople puckering coordinates as collective variables. The computed FEL supports only two of the previously proposed itineraries, 2SO → [2,5B]ǂ → 5S1 and 1S3 → [4H3]ǂ → 4C1, which clearly appear in low energy regions of the FEL. Consistently, 2SO and 1S3 are conformations preactivated for catalysis in terms of free energy/anomeric charge and bond distances. The results however exclude the OE → [OS2]ǂ → B2,5 itinerary that has been recently proposed for a family 11 xylanase. Classical and ab initio QM/MM molecular dynamics simulations reveal that, in this case, the observed OE conformation has been enforced by enzyme mutation. These results add a word of caution on using modified enzymes to inform on catalytic conformational itineraries of glycoside hydrolases. PMID:29560204

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

A complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis.

PubMed

Jones, Hannah B L; Wells, Stephen A; Prentice, Erica J; Kwok, Anthony; Liang, Liyin L; Arcus, Vickery L; Pudney, Christopher R

2017-09-01

Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well-defined static conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called 'conformational sampling' involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach. © 2017 Federation of European Biochemical Societies.

Conformational Sub-states and Populations in Enzyme Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, Pratul K; Doucet, Nicholas; Chennubholta, C

reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surroundingmore » environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.« less

In silico strategies toward enzyme function and dynamics.

PubMed

Estácio, Sílvia G

2012-01-01

Enzymes are outstanding biocatalysts involved in a plethora of chemical reactions occurring in the cell. Despite their incommensurable importance, a comprehensive understanding of enzyme catalysis is still missing. This task becomes more laborious given the unavoidability of including the inherent dynamic nature of enzymes into that description. As such, it is essential to ascertain the nature and contribution of enzyme conformational changes to catalysis and to evaluate the adequacy of the proposal associating protein internal motions to the rate enhancement achieved. Dynamic events in enzymes span a wide range of time- and length-scales which have led to a surge in multiscale methodologies targeting enzyme function and dynamics. Computational strategies assume a preponderant role in such studies by allowing the atomic detail investigation of the fundamental mechanisms of enzyme catalysis thus surpassing what is achievable through experiments. While high-accuracy quantum mechanical methods are indicated to uncover the details of the chemical reaction occurring at the active site, molecular mechanical force fields and molecular dynamics approaches provide powerful means to access the conformational energy landscape accessible to enzymes. This review outlines some of the most important in silico methodologies in this area, highlighting examples of problems tackled and the insights obtained. Copyright © 2012 Elsevier Inc. All rights reserved.

Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis

PubMed Central

Parks, Joseph W.; Kappel, Kalli; Das, Rhiju; Stone, Michael D.

2017-01-01

Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation. PMID:28096444

The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics/Molecular Mechanics Simulations

PubMed Central

Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

2012-01-01

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and Pi in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and Pi release back to the diphosphate. Furthermore, the Ras·Mg2+ complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. PMID:22853907

Elucidation of the conformational free energy landscape in H.pylori LuxS and its implications to catalysis.

PubMed

Bhattacharyya, Moitrayee; Vishveshwara, Saraswathi

2010-08-12

One of the major challenges in understanding enzyme catalysis is to identify the different conformations and their populations at detailed molecular level in response to ligand binding/environment. A detail description of the ligand induced conformational changes provides meaningful insights into the mechanism of action of enzymes and thus its function. In this study, we have explored the ligand induced conformational changes in H.pylori LuxS and the associated mechanistic features. LuxS, a dimeric protein, produces the precursor (4,5-dihydroxy-2,3-pentanedione) for autoinducer-2 production which is a signalling molecule for bacterial quorum sensing. We have performed molecular dynamics simulations on H.pylori LuxS in its various ligand bound forms and analyzed the simulation trajectories using various techniques including the structure network analysis, free energy evaluation and water dynamics at the active site. The results bring out the mechanistic details such as co-operativity and asymmetry between the two subunits, subtle changes in the conformation as a response to the binding of active and inactive forms of ligands and the population distribution of different conformations in equilibrium. These investigations have enabled us to probe the free energy landscape and identify the corresponding conformations in terms of network parameters. In addition, we have also elucidated the variations in the dynamics of water co-ordination to the Zn2+ ion in LuxS and its relation to the rigidity at the active sites. In this article, we provide details of a novel method for the identification of conformational changes in the different ligand bound states of the protein, evaluation of ligand-induced free energy changes and the biological relevance of our results in the context of LuxS structure-function. The methodology outlined here is highly generalized to illuminate the linkage between structure and function in any protein of known structure.

Methods for Efficiently and Accurately Computing Quantum Mechanical Free Energies for Enzyme Catalysis.

PubMed

Kearns, F L; Hudson, P S; Boresch, S; Woodcock, H L

2016-01-01

Enzyme activity is inherently linked to free energies of transition states, ligand binding, protonation/deprotonation, etc.; these free energies, and thus enzyme function, can be affected by residue mutations, allosterically induced conformational changes, and much more. Therefore, being able to predict free energies associated with enzymatic processes is critical to understanding and predicting their function. Free energy simulation (FES) has historically been a computational challenge as it requires both the accurate description of inter- and intramolecular interactions and adequate sampling of all relevant conformational degrees of freedom. The hybrid quantum mechanical molecular mechanical (QM/MM) framework is the current tool of choice when accurate computations of macromolecular systems are essential. Unfortunately, robust and efficient approaches that employ the high levels of computational theory needed to accurately describe many reactive processes (ie, ab initio, DFT), while also including explicit solvation effects and accounting for extensive conformational sampling are essentially nonexistent. In this chapter, we will give a brief overview of two recently developed methods that mitigate several major challenges associated with QM/MM FES: the QM non-Boltzmann Bennett's acceptance ratio method and the QM nonequilibrium work method. We will also describe usage of these methods to calculate free energies associated with (1) relative properties and (2) along reaction paths, using simple test cases with relevance to enzymes examples. © 2016 Elsevier Inc. All rights reserved.

The Ligand Trans Influence Governs Conformation in Cobalamin-Dependent Methionine Synthase†

PubMed Central

Fleischhacker, Angela S.; Matthews, Rowena G.

2008-01-01

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a large, modular enzyme that uses a cobalamin prosthetic group as a donor or acceptor in three separate methyl transfer reactions. The prosthetic group alternates between methylcobalamin and cob(I)alamin during catalysis as homocysteine is converted to methionine using a methyl group derived from methyltetrahydrofolate. Occasional oxidation of cob(I)alamin to cob(II)alamin inactivates the enzyme. Reductive methylation with flavodoxin and adenosylmethionine returns the enzyme to an active methylcobalamin state. At different points during the reaction cycle, the coordination of the cobalt of the cobalamin changes. The imidazole side chain of His759 coordinates to cobalamin in a “His-on” state and dissociates to produce a “His-off” state. The His-off state has been associated with a conformation of MetH that is poised for reactivation of cobalamin by reductive methylation rather than catalysis. Our studies on cob(III)alamins bound to MetH, specifically aqua-, methyl-, and n-propylcobalamin, show a correlation between the accessibility of the reactivation conformation and the order of the established ligand trans influence. The trans influence also controls the affinity of MetH in the cob(III)alamin form for flavodoxin. Flavodoxin, which acts to shift the conformational equilibrium towards the reactivation conformation, binds less tightly to MetH when the cob(III)alamin has a strong trans ligand and therefore has less positive charge on cobalt. These results are compared to those for cob(II)alamin MetH, illustrating that access to the reactivation conformation is governed by the net charge on the cobalt as well as the trans influence in cob(III)alamins. PMID:17924667

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner*

PubMed Central

Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.

2015-01-01

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase

PubMed Central

Bae, Sung-Hun; Duggan, Brendan M.; Benkovic, Stephen J.; Dyson, H. Jane; Wright, Peter E

2013-01-01

Correlated networks of amino acids have been proposed to play a fundamental role in allostery and enzyme catalysis. These networks of amino acids can be traced from surface-exposed residues all the way into the active site, and disruption of these networks can decrease enzyme activity. Substitution of the distal Gly121 residue in E.coli dihydrofolate reductase results in up to a 200-fold decrease in the hydride transfer rate despite the fact that the residue is located 15 Å from the active-site center. In the present study, NMR relaxation experiments are used to demonstrate that dynamics on the ps-ns and μs-ms timescales are changed significantly in the G121V mutant of dihydrofolate reductase. In particular, ps-ns timescale dynamics are decreased in the FG loop (containing the mutated residue 121) and the neighboring active-site loop (the Met20 loop) in the mutant compared to wild-type enzyme, suggesting that these loops are dynamically coupled. Changes in methyl order parameters reveal a pathway by which dynamic perturbations can be propagated more than 25 Å across the protein from the site of mutation. All of the enzyme complexes, including the model Michaelis complex with folate and NADP+ bound, assume an occluded ground state conformation, and we do not observe sampling of a higher energy closed conformation by 15N R2 relaxation dispersion. This is highly significant, since it is only in the closed conformation that the cofactor and substrate reactive centers are positioned for reaction. The mutation also impairs μs - ms timescale fluctuations that have been implicated in product release from the wild type enzyme. Our results are consistent with an important role for Gly121 in controlling protein dynamics critical for enzyme function and further validate the dynamic energy landscape hypothesis of enzyme catalysis. PMID:23758161

Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

DOE PAGES

Deng, Xiangyu; Qin, Xiangjing; Chen, Lei; ...

2016-01-21

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunitmore » fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).« less

Quantum clustering and network analysis of MD simulation trajectories to probe the conformational ensembles of protein-ligand interactions.

PubMed

Bhattacharyya, Moitrayee; Vishveshwara, Saraswathi

2011-07-01

In this article, we present a novel application of a quantum clustering (QC) technique to objectively cluster the conformations, sampled by molecular dynamics simulations performed on different ligand bound structures of the protein. We further portray each conformational population in terms of dynamically stable network parameters which beautifully capture the ligand induced variations in the ensemble in atomistic detail. The conformational populations thus identified by the QC method and verified by network parameters are evaluated for different ligand bound states of the protein pyrrolysyl-tRNA synthetase (DhPylRS) from D. hafniense. The ligand/environment induced re-distribution of protein conformational ensembles forms the basis for understanding several important biological phenomena such as allostery and enzyme catalysis. The atomistic level characterization of each population in the conformational ensemble in terms of the re-orchestrated networks of amino acids is a challenging problem, especially when the changes are minimal at the backbone level. Here we demonstrate that the QC method is sensitive to such subtle changes and is able to cluster MD snapshots which are similar at the side-chain interaction level. Although we have applied these methods on simulation trajectories of a modest time scale (20 ns each), we emphasize that our methodology provides a general approach towards an objective clustering of large-scale MD simulation data and may be applied to probe multistate equilibria at higher time scales, and to problems related to protein folding for any protein or protein-protein/RNA/DNA complex of interest with a known structure.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Optimization of the in silico designed Kemp eliminase KE70 by computational design and directed evolution

PubMed Central

Khersonsky, Olga; Röthlisberger, Daniela; Wollacott, Andrew M.; Murphy, Paul; Dym, Orly; Albeck, Shira; Kiss, Gert; Houk, K. N.; Baker, David; Tawfik, Dan S.

2013-01-01

Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency, and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher kcat and lower KM values, with the best variants exhibiting kcat/KM values of >5x104 s−1M−1. The optimized KE70 variants were characterized structurally and biochemically providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active site cavity to achieve tighter substrate binding; second, the fine-tuning of the electrostatics around the catalytic His-Asp dyad; and third, stabilization of the active-site dyad in a conformation optimal for catalysis. PMID:21277311

Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

PubMed Central

2015-01-01

The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

PubMed Central

2012-01-01

Background During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation. PMID:22676913

Hydrogen tunneling links protein dynamics to enzyme catalysis.

PubMed

Klinman, Judith P; Kohen, Amnon

2013-01-01

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis

PubMed Central

Klinman, Judith P.; Kohen, Amnon

2014-01-01

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C–H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial. PMID:23746260

The energy landscape of adenylate kinase during catalysis

PubMed Central

Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

2014-01-01

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site. PMID:25580578

The CoFactor database: organic cofactors in enzyme catalysis.

PubMed

Fischer, Julia D; Holliday, Gemma L; Thornton, Janet M

2010-10-01

Organic enzyme cofactors are involved in many enzyme reactions. Therefore, the analysis of cofactors is crucial to gain a better understanding of enzyme catalysis. To aid this, we have created the CoFactor database. CoFactor provides a web interface to access hand-curated data extracted from the literature on organic enzyme cofactors in biocatalysis, as well as automatically collected information. CoFactor includes information on the conformational and solvent accessibility variation of the enzyme-bound cofactors, as well as mechanistic and structural information about the hosting enzymes. The database is publicly available and can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/CoFactor.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode.

PubMed

Chen, Yaozong; Li, Tin Lok; Lin, Xingbang; Li, Xin; Li, Xiang David; Guo, Zhihong

2017-07-21

o -Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Probing the electrostatics of active site microenvironments along the catalytic cycle for Escherichia coli dihydrofolate reductase.

PubMed

Liu, C Tony; Layfield, Joshua P; Stewart, Robert J; French, Jarrod B; Hanoian, Philip; Asbury, John B; Hammes-Schiffer, Sharon; Benkovic, Stephen J

2014-07-23

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and (13)C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor-acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR.

Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

PubMed Central

2015-01-01

Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecDHFR. PMID:24977791

Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis

PubMed Central

Lee, Tai-Sung; Wong, Kin-Yiu; Giambasu, George M.; York, Darrin M.

2016-01-01

Herein we summarize our progress toward the understanding of hammerhead ribozyme (HHR) catalysis through a multiscale simulation strategy. Simulation results collectively paint a picture of HHR catalysis: HHR first folds to form an electronegative active site pocket to recruit a threshold occupation of cationic charges, either a Mg2+ ion or multiple monovalent cations. Catalytically active conformations that have good in-line fitness are supported by specific metal ion coordination patterns that involve either a bridging Mg2+ ion or multiple Na+ ions, one of which is also in a bridging coordination pattern. In the case of a single Mg2+ ion bound in the active site, the Mg2+ ion undergoes a migration that is coupled with deprotonation of the nucleophile (C17:O2′). As the reaction proceeds, the Mg2+ ion stabilizes the accumulating charge of the leaving group and significantly increases the general acid ability of G8:O2′. Further computational mutagenesis simulations suggest that the disruptions due to mutations may severely impact HHR catalysis at different stages of the reaction. Catalytic mechanisms supported by the simulation results are consistent with available structural and biochemical experiments, and together they advance our understanding of HHR catalysis. PMID:24156941

13C ENDOR Spectroscopy of Lipoxygenase-Substrate Complexes Reveals the Structural Basis for C-H Activation by Tunneling.

PubMed

Horitani, Masaki; Offenbacher, Adam R; Carr, Cody A Marcus; Yu, Tao; Hoeke, Veronika; Cutsail, George E; Hammes-Schiffer, Sharon; Klinman, Judith P; Hoffman, Brian M

2017-02-08

In enzymatic C-H activation by hydrogen tunneling, reduced barrier width is important for efficient hydrogen wave function overlap during catalysis. For native enzymes displaying nonadiabatic tunneling, the dominant reactive hydrogen donor-acceptor distance (DAD) is typically ca. 2.7 Å, considerably shorter than normal van der Waals distances. Without a ground state substrate-bound structure for the prototypical nonadiabatic tunneling system, soybean lipoxygenase (SLO), it has remained unclear whether the requisite close tunneling distance occurs through an unusual ground state active site arrangement or by thermally sampling conformational substates. Herein, we introduce Mn 2+ as a spin-probe surrogate for the SLO Fe ion; X-ray diffraction shows Mn-SLO is structurally faithful to the native enzyme. 13 C ENDOR then reveals the locations of 13 C10 and reactive 13 C11 of linoleic acid relative to the metal; 1 H ENDOR and molecular dynamics simulations of the fully solvated SLO model using ENDOR-derived restraints give additional metrical information. The resulting three-dimensional representation of the SLO active site ground state contains a reactive (a) conformer with hydrogen DAD of ∼3.1 Å, approximately van der Waals contact, plus an inactive (b) conformer with even longer DAD, establishing that stochastic conformational sampling is required to achieve reactive tunneling geometries. Tunneling-impaired SLO variants show increased DADs and variations in substrate positioning and rigidity, confirming previous kinetic and theoretical predictions of such behavior. Overall, this investigation highlights the (i) predictive power of nonadiabatic quantum treatments of proton-coupled electron transfer in SLO and (ii) sensitivity of ENDOR probes to test, detect, and corroborate kinetically predicted trends in active site reactivity and to reveal unexpected features of active site architecture.

Pyranopterin conformation defines the function of molybdenum and tungsten enzymes.

PubMed

Rothery, Richard A; Stein, Benjamin; Solomonson, Matthew; Kirk, Martin L; Weiner, Joel H

2012-09-11

We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state. Enzymes belonging to the bacterial dimethyl sulfoxide reductase (DMSOR) family contain a metal-bis-pyranopterin cofactor, the two pyranopterins of which have distinct conformations, with one similar to the predicted tetrahydro form, and the other similar to the predicted dihydro form. Enzymes containing a single pyranopterin belong to either the xanthine dehydrogenase (XDH) or sulfite oxidase (SUOX) families, and these have pyranopterin conformations similar to those predicted for tetrahydro and dihydro forms, respectively. This work provides keen insight into the roles of pyranopterin conformation and oxidation state in catalysis, redox potential modulation of the metal site, and catalytic function.

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

PubMed Central

Gabba, Matteo; Poblete, Simón; Rosenkranz, Tobias; Katranidis, Alexandros; Kempe, Daryan; Züchner, Tina; Winkler, Roland G.; Gompper, Gerhard; Fitter, Jörg

2014-01-01

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions. PMID:25418172

Structured crowding and its effects on enzyme catalysis.

PubMed

Ma, Buyong; Nussinov, Ruth

2013-01-01

Macromolecular crowding decreases the diffusion rate, shifts the equilibrium of protein-protein and protein-substrate interactions, and changes protein conformational dynamics. Collectively, these effects contribute to enzyme catalysis. Here we describe how crowding may bias the conformational change and dynamics of enzyme populations and in this way affect catalysis. Crowding effects have been studied using artificial crowding agents and in vivo-like environments. These studies revealed a correlation between protein dynamics and function in the crowded environment. We suggest that crowded environments be classified into uniform crowding and structured crowding. Uniform crowding represents random crowding conditions created by synthetic particles with a narrow size distribution. Structured crowding refers to the highly coordinated cellular environment, where proteins and other macromolecules are clustered and organized. In structured crowded environments the perturbation of protein thermal stability may be lower; however, it may still be able to modulate functions effectively and dynamically. Dynamic, allosteric enzymes could be more sensitive to cellular perturbations if their free energy landscape is flatter around the native state; on the other hand, if their free energy landscape is rougher, with high kinetic barriers separating deep minima, they could be more robust. Above all, cells are structured; and this holds both for the cytosol and for the membrane environment. The crowded environment is organized, which limits the search, and the crowders are not necessarily inert. More likely, they too transmit allosteric effects, and as such play important functional roles. Overall, structured cellular crowding may lead to higher enzyme efficiency and specificity.

Adenylosuccinate synthetase: recent developments.

PubMed

Honzatko, R B; Stayton, M M; Fromm, H J

1999-01-01

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

The interactions in the carboxyl terminus of human 4-hydroxyphenylpyruvate dioxygenase are critical to mediate the conformation of the final helix and the tail to shield the active site for catalysis.

PubMed

Lin, Jang-Foung; Sheih, Yung-Lin; Chang, Tsu-Chung; Chang, Ni-Yuan; Chang, Chiung-Wen; Shen, Chia-Pei; Lee, Hwei-Jen

2013-01-01

4-Hydroxylphenylpyruvate dioxygenase (4-HPPD) is an important enzyme for tyrosine catabolism, which catalyzes the conversion of 4-hydroxylphenylpyruvate (4-HPP) to homogentisate. In the present study, human 4-HPPD was cloned and expressed in E. coli. The kinetic parameters for 4-HPP conversion were: k cat=2.2 ± 0.1 s(-1); and K m=0.08 ± 0.02 mM. Sequence alignments show that human 4-HPPD possesses an extended C-terminus compared to other 4-HPPD enzymes. Successive truncation of the disordered tail which follows the final α-helix resulted in no changes in the K m value for 4-HPP substrate but the k cat values were significantly reduced. The results suggest that this disordered C-terminal tail plays an important role in catalysis. For inspection the effect of terminal truncation on protein structure, mutant models were built. These models suggest that the different conformation of E254, R378 and Q375 in the final helix might be the cause of the activity loss. In the structure E254 interacts with R378, the end residue in the final helix; mutation of either one of these residues causes a ca. 95% reductions in k cat values. Q375 provides bifurcate interactions to fix the tail and the final helix in position. The model of the Q375N mutant shows that a solvent accessible channel opens to the putative substrate binding site, suggesting this is responsible for the complete loss of activity. These results highlight the critical role of Q375 in orientating the tail and ensuring the conformation of the terminal α-helix to maintain the integrity of the active site for catalysis.

Asymmetric Catalysis with bis(hydroxyphenyl)diamides/rare-earth metal complexes.

PubMed

Kumagai, Naoya; Shibasaki, Masakatsu

2013-01-02

A series of asymmetric catalysts composed of conformationally flexible amide-based chiral ligands and rare-earth metals was developed for proton-transfer catalysis. These ligands derived from amino acids provide an intriguing chiral platform for the formation of asymmetric catalysts upon complexation with rare-earth metals. The scope of this arsenal of catalysts was further broadened by the development of heterobimetallic catalytic systems. The cooperative function of hydrogen bonding and metal coordination resulted in intriguing substrate specificity and stereocontrol, and the dynamic nature of the catalysts led to a switch of their function. Herein, we summarize our recent exploration of this class of catalysts. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis.

PubMed

Burschowsky, Daniel; van Eerde, André; Ökvist, Mats; Kienhöfer, Alexander; Kast, Peter; Hilvert, Donald; Krengel, Ute

2014-12-09

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandao, T.; Robinson, H; Johnson, S

Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presencemore » of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.« less

Surfactant-assisted stabilization of Au colloids on solids for heterogeneous catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhan, Wangcheng; Shu, Yuan; Sheng, Yujie

Here, the stabilization of surfactant-assisted synthesized colloidal noble metal nanoparticles (NPs, e.g., Au NPs) on solids is a promising strategy for preparing supported nanocatalysts for heterogeneous catalysis because of their uniform particle sizes, controllable shapes, and tunable compositions. However, the removal of surfactants to obtain clean surfaces for catalysis through traditional approaches (e.g., solvent extraction and thermal decomposition) can easily induce the sintering of NPs, greatly hampering their use in synthesis of novel catalysts. Herein, we demonstrate that such unwanted surfactants can be utilized to stabilize NPs on solids via a simple yet efficient thermal annealing strategy. After being annealedmore » in N 2 flow, the surface-bound surfactants are in situ carbonized as sacrificial architectures that form a conformal coating on NPs and assist in creating an enhanced metal-support interaction between NPs and substrate, thus slowing down the Ostwald ripening process during post-oxidative calcination to remove surface covers.« less

Surfactant-assisted stabilization of Au colloids on solids for heterogeneous catalysis

DOE PAGES

Zhan, Wangcheng; Shu, Yuan; Sheng, Yujie; ...

2017-03-22

Here, the stabilization of surfactant-assisted synthesized colloidal noble metal nanoparticles (NPs, e.g., Au NPs) on solids is a promising strategy for preparing supported nanocatalysts for heterogeneous catalysis because of their uniform particle sizes, controllable shapes, and tunable compositions. However, the removal of surfactants to obtain clean surfaces for catalysis through traditional approaches (e.g., solvent extraction and thermal decomposition) can easily induce the sintering of NPs, greatly hampering their use in synthesis of novel catalysts. Herein, we demonstrate that such unwanted surfactants can be utilized to stabilize NPs on solids via a simple yet efficient thermal annealing strategy. After being annealedmore » in N 2 flow, the surface-bound surfactants are in situ carbonized as sacrificial architectures that form a conformal coating on NPs and assist in creating an enhanced metal-support interaction between NPs and substrate, thus slowing down the Ostwald ripening process during post-oxidative calcination to remove surface covers.« less

Protein dynamics and enzyme catalysis: insights from simulations.

PubMed

McGeagh, John D; Ranaghan, Kara E; Mulholland, Adrian J

2011-08-01

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist - or student - to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches. Copyright © 2010 Elsevier B.V. All rights reserved.

The energy landscape of adenylate kinase during catalysis

DOE PAGES

Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; ...

2015-01-12

Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. In this paper, we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg 2+ cofactor activates two distinct molecular events: phosphoryl transfer (>10 5-fold) and lid opening (10 3-fold). In contrast, mutation of an essential active site arginine decelerates phosphorylmore » transfer 10 3-fold without substantially affecting lid opening. Finally, our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.« less

Conformational Flexibility of Metazoan Fatty Acid Synthase Enables Catalysis

PubMed Central

Brignole, Edward J.; Smith, Stuart; Asturias, Francisco J.

2008-01-01

The metazoan cytosolic fatty acid synthase (FAS) contains all of the enzymes required for de novo fatty acid biosynthesis covalently linked around two reaction chambers. While the 3D architecture of FAS has been mostly defined, it is unclear how reaction intermediates can transfer between distant catalytic domains. Using single-particle electron microscopy we have identified a near continuum of conformations consistent with remarkable flexibility of FAS. The distribution of conformations was influenced by the presence of substrates and altered by different catalytic mutations suggesting a direct correlation between conformation and specific enzymatic activities. 3D reconstructions were interpreted by docking high-resolution structures of individual domains and illustrate that the substrate loading and condensation domains dramatically swing and swivel to access substrates within either reaction chamber. Concomitant rearrangement of the β-carbon processing domains synchronizes acyl-chain reduction in one chamber with acyl-chain elongation in the other. PMID:19151726

Origin of fast catalysis in allylic amination reactions catalyzed by Pd-Ti heterobimetallic complexes.

PubMed

Walker, Whitney K; Kay, Benjamin M; Michaelis, Scott A; Anderson, Diana L; Smith, Stacey J; Ess, Daniel H; Michaelis, David J

2015-06-17

Experiments and density functional calculations were used to quantify the impact of the Pd-Ti interaction in the cationic heterobimetallic Cl2Ti(N(t)BuPPh2)2Pd(η(3)-methallyl) catalyst 1 used for allylic aminations. The catalytic significance of the Pd-Ti interaction was evaluated computationally by examining the catalytic cycle for catalyst 1 with a conformation where the Pd-Ti interaction is intact versus one where the Pd-Ti interaction is severed. Studies were also performed on the relative reactivity of the cationic monometallic (CH2)2(N(t)BuPPh2)2Pd(η(3)-methallyl) catalyst 2 where the Ti from catalyst 1 was replaced by an ethylene group. These computational and experimental studies revealed that the Pd-Ti interaction lowers the activation barrier for turnover-limiting amine reductive addition and accelerates catalysis up to 10(5). The Pd-Ti distance in 1 is the result of the N(t)Bu groups enforcing a boat conformation that brings the two metals into close proximity, especially in the transition state. The turnover frequency of classic Pd π allyl complexes was compared to that of 1 to determine the impact of P-Pd-P coordination angle and ligand electronic properties on catalysis. These experiments identified that cationic (PPh3)2Pd(η(3)-CH2C(CH3)CH2) catalyst 3 performs similarly to 1 for allylic aminations with diethylamine. However, computations and experiment reveal that the apparent similarity in reactivity is due to very fast reaction kinetics. The higher reactivity of 1 versus 3 was confirmed in the reaction of methallyl chloride and 2,2,6,6-tetramethylpiperidine (TMP). Overall, experiments and calculations demonstrate that the Pd-Ti interaction induces and is responsible for significantly lower barriers and faster catalysis for allylic aminations.

Conformational Changes in Orotidine 5-Monophosphate Decarboxylase: "Remote" Residues That Stabilize the Active Conformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wood, B.; Amyes, T; Fedorov, A

2010-01-01

The structural factors responsible for the extraordinary rate enhancement ({approx}10{sup 17}) of the reaction catalyzed by orotidine 5{prime}-monophosphate decarboxylase (OMPDC) have not been defined. Catalysis requires a conformational change that closes an active site loop and 'clamps' the orotate base proximal to hydrogen-bonded networks that destabilize the substrate and stabilize the intermediate. In the OMPDC from Methanobacter thermoautotrophicus, a 'remote' structurally conserved cluster of hydrophobic residues that includes Val 182 in the active site loop is assembled in the closed, catalytically active conformation. Substitution of these residues with Ala decreases k{sub cat}/K{sub m} with a minimal effect on k{sub cat},more » providing evidence that the cluster stabilizes the closed conformation. The intrinsic binding energies of the 5{prime}-phosphate group of orotidine 5{prime}-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion.« less

A surprising role for conformational entropy in protein function

PubMed Central

Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

2014-01-01

Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, Sarah M.; Holyoak, Todd

2008-09-17

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less

Enzymes With Lid-Gated Active Sites Must Operate By An Induced Fit Mechanism Instead of Conformational Selection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, S.M.; Holyoak, T.

2009-05-26

The induced fit and conformational selection/population shift models are two extreme cases of a continuum aimed at understanding the mechanism by which the final key-lock or active enzyme conformation is achieved upon formation of the correctly ligated enzyme. Structures of complexes representing the Michaelis and enolate intermediate complexes of the reaction catalyzed by phosphoenolpyruvate carboxykinase provide direct structural evidence for the encounter complex that is intrinsic to the induced fit model and not required by the conformational selection model. In addition, the structural data demonstrate that the conformational selection model is not sufficient to explain the correlation between dynamics andmore » catalysis in phosphoenolpyruvate carboxykinase and other enzymes in which the transition between the uninduced and the induced conformations occludes the active site from the solvent. The structural data are consistent with a model in that the energy input from substrate association results in changes in the free energy landscape for the protein, allowing for structural transitions along an induced fit pathway.« less

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

PubMed Central

Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

2007-01-01

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

Shift in the equilibrium between on and off states of the allosteric switch in Ras-GppNHp affected by small molecules and bulk solvent composition.

PubMed

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-07

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

PubMed

Sheng, Yinghong; Zhong, Linghao; Guo, Dahai; Lau, Gavin; Feng, Changjian

2015-12-01

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production. Copyright © 2015 Elsevier Inc. All rights reserved.

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-31

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) canmore » selectively shift the equilibrium to the 'on' state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the 'ordered off' state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-{beta}. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.« less

Attractive noncovalent interactions in asymmetric catalysis: Links between enzymes and small molecule catalysts

PubMed Central

Knowles, Robert R.; Jacobsen, Eric N.

2010-01-01

Catalysis by neutral, organic, small molecules capable of binding and activating substrates solely via noncovalent interactions—particularly H-bonding—has emerged as an important approach in organocatalysis. The mechanisms by which such small molecule catalysts induce high enantioselectivity may be quite different from those used by catalysts that rely on covalent interactions with substrates. Attractive noncovalent interactions are weaker, less distance dependent, less directional, and more affected by entropy than covalent interactions. However, the conformational constraint required for high stereoinduction may be achieved, in principle, if multiple noncovalent attractive interactions are operating in concert. This perspective will outline some recent efforts to elucidate the cooperative mechanisms responsible for stereoinduction in highly enantioselective reactions promoted by noncovalent catalysts. PMID:20956302

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

PubMed

Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

2012-10-19

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities.

PubMed

Neumann, Piotr; Tittmann, Kai

2014-12-01

Although general principles of enzyme catalysis are fairly well understood nowadays, many important details of how exactly the substrate is bound and processed in an enzyme remain often invisible and as such elusive. In fortunate cases, structural analysis of enzymes can be accomplished at true atomic resolution thus making possible to shed light on otherwise concealed fine-structural traits of bound substrates, intermediates, cofactors and protein groups. We highlight recent structural studies of enzymes using ultrahigh-resolution X-ray protein crystallography showcasing its enormous potential as a tool in the elucidation of enzymatic mechanisms and in unveiling fundamental principles of enzyme catalysis. We discuss the observation of seemingly hyper-reactive, physically distorted cofactors and intermediates with elongated scissile substrate bonds, the detection of 'hidden' conformational and chemical equilibria and the analysis of protonation states with surprising findings. In delicate cases, atomic resolution is required to unambiguously disclose the identity of atoms as demonstrated for the metal cluster in nitrogenase. In addition to the pivotal structural findings and the implications for our understanding of enzyme catalysis, we further provide a practical framework for resolution enhancement through optimized data acquisition and processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Perspectives on electrostatics and conformational motions in enzyme catalysis.

PubMed

Hanoian, Philip; Liu, C Tony; Hammes-Schiffer, Sharon; Benkovic, Stephen

2015-02-17

CONSPECTUS: Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors.

Perspectives on Electrostatics and Conformational Motions in Enzyme Catalysis

PubMed Central

2016-01-01

Conspectus Enzymes are essential for all living organisms, and their effectiveness as chemical catalysts has driven more than a half century of research seeking to understand the enormous rate enhancements they provide. Nevertheless, a complete understanding of the factors that govern the rate enhancements and selectivities of enzymes remains elusive, due to the extraordinary complexity and cooperativity that are the hallmarks of these biomolecules. We have used a combination of site-directed mutagenesis, pre-steady-state kinetics, X-ray crystallography, nuclear magnetic resonance (NMR), vibrational and fluorescence spectroscopies, resonance energy transfer, and computer simulations to study the implications of conformational motions and electrostatic interactions on enzyme catalysis in the enzyme dihydrofolate reductase (DHFR). We have demonstrated that modest equilibrium conformational changes are functionally related to the hydride transfer reaction. Results obtained for mutant DHFRs illustrated that reductions in hydride transfer rates are correlated with altered conformational motions, and analysis of the evolutionary history of DHFR indicated that mutations appear to have occurred to preserve both the hydride transfer rate and the associated conformational changes. More recent results suggested that differences in local electrostatic environments contribute to finely tuning the substrate pKa in the initial protonation step. Using a combination of primary and solvent kinetic isotope effects, we demonstrated that the reaction mechanism is consistent across a broad pH range, and computer simulations suggested that deprotonation of the active site Tyr100 may play a crucial role in substrate protonation at high pH. Site-specific incorporation of vibrational thiocyanate probes into the ecDHFR active site provided an experimental tool for interrogating these microenvironments and for investigating changes in electrostatics along the DHFR catalytic cycle. Complementary molecular dynamics simulations in conjunction with mixed quantum mechanical/molecular mechanical calculations accurately reproduced the vibrational frequency shifts in these probes and provided atomic-level insight into the residues influencing these changes. Our findings indicate that conformational and electrostatic changes are intimately related and functionally essential. This approach can be readily extended to the study of other enzyme systems to identify more general trends in the relationship between conformational fluctuations and electrostatic interactions. These results are relevant to researchers seeking to design novel enzymes as well as those seeking to develop therapeutic agents that function as enzyme inhibitors. PMID:25565178

Multiscale methods for computational RNA enzymology

PubMed Central

Panteva, Maria T.; Dissanayake, Thakshila; Chen, Haoyuan; Radak, Brian K.; Kuechler, Erich R.; Giambaşu, George M.; Lee, Tai-Sung; York, Darrin M.

2016-01-01

RNA catalysis is of fundamental importance to biology and yet remains ill-understood due to its complex nature. The multi-dimensional “problem space” of RNA catalysis includes both local and global conformational rearrangements, changes in the ion atmosphere around nucleic acids and metal ion binding, dependence on potentially correlated protonation states of key residues and bond breaking/forming in the chemical steps of the reaction. The goal of this article is to summarize and apply multiscale modeling methods in an effort to target the different parts of the RNA catalysis problem space while also addressing the limitations and pitfalls of these methods. Classical molecular dynamics (MD) simulations, reference interaction site model (RISM) calculations, constant pH molecular dynamics (CpHMD) simulations, Hamiltonian replica exchange molecular dynamics (HREMD) and quantum mechanical/molecular mechanical (QM/MM) simulations will be discussed in the context of the study of RNA backbone cleavage transesterification. This reaction is catalyzed by both RNA and protein enzymes, and here we examine the different mechanistic strategies taken by the hepatitis delta virus ribozyme (HDVr) and RNase A. PMID:25726472

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

PubMed Central

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine

PubMed Central

Ouzon-Shubeita, Hala; Lee, Seongmin

2014-01-01

N7-Methyl-2′-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2′-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2′-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson–Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate. PMID:24966350

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

Enzyme activation through the utilization of intrinsic dianion binding energy.

PubMed

Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

2017-03-01

We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Active site CP-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

PubMed

Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

2018-04-01

Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (C P ) located in a conserved sequence Pxxx(T/S)xxC P motif known as C P -loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the C P -loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure. Despite many studies, little is known about the precise structural and dynamic roles of the C P -loop on Prxs functions. Herein, the comprehensive biochemical and biophysical studies on Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) and the C P -loop mutants, EcAhpC-F45A and EcAhpC-F45P reveal that the reduced form of the C P -loop adopts conformational dynamics, which is essential for effective peroxide reduction. Furthermore, the point mutants alter the structure and dynamics of the reduced form of the C P -loop and, thereby, affect substrate binding, catalysis, oligomerization, stability and overoxidiation. In the oxidized form, due to restricted C P -loop dynamics, the EcAhpC-F45P mutant favours a decamer formation, which enhances the effective recycling by physiological reductases compared to wild-type EcAhpC. In addition, the study reveals that residue F45 increases the specificity of Prxs-reductase interactions. Based on these studies, we propose an evolution of the C P -loop with confined sequence conservation within Prxs subfamilies that might optimize the functional adaptation of Prxs into various physiological roles. Copyright © 2018 Elsevier Inc. All rights reserved.

Yeast enolase: mechanism of activation by metal ions.

PubMed

Brewer, J M

1981-01-01

Yeast enolase as prepared by current procedures is inherently chemically homogeneous, though deamidation and partial denaturation can produce electrophoretically distinct forms. A true isozyme of the enzyme exists but does not survive the purification procedure. The chemical sequence for both has been established. The enzyme behaves in solution like a compact, nearly spherical molecule of moderate hydration. Strong intramolecular forces maintain the structure of the individual subunits. The enzyme as isolated is dimeric. If dissociated in the presence of magnesium ions and substrate, then the subunits are active, but if the dissociation occurs in the absence of metal ions, they are inactive until they have reassociated and undergone a first order "annealing" process. Magnesium (II) enhances association. The interaction between the subunits is hydrophobic in character. The enzyme can bind up to 2 mol of most metal ions in "conformational" sites which then allows up to 2 mol of substrate or some substrate analogue to bind. This is not sufficient for catalysis, but conformational metal ions do more than just allow substrate binding. A change in the environment of the metal ions occurs on substrate or substrate analogue binding. There is an absolute correlation between the occurrence of a structural change undergone by the 3-amino analogue of phosphoenolpyruvate and whether the metal ions produce any level of enzymatic activity. For catalysis, two more moles of metal ions, called "catalytic", must bind. There is evidence that the enzymatic reaction involves a carbanion mechanism. It is likely that two more moles of metal ion can bind which inhibit the reaction. The requirement for 2 mol of metal ion per subunit which contribute in different ways to catalysis is exhibited by a number of other enzymes.

COBALAMIN- AND COBAMIDE-DEPENDENT METHYLTRANSFERASES

PubMed Central

Matthews, Rowena G.; Koutmos, Markos; Datta, Supratim

2008-01-01

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple SN2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co+1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled. PMID:19059104

Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with Alpha-Alpha Domain Architecture that Catalyzes a Unique Cyclization-Fragmentation Reaction Sequence

PubMed Central

Harris, Golda G.; Lombardi, Patrick M.; Pemberton, Travis A.; Matsui, Tsutomu; Weiss, Thomas M.; Cole, Kathryn E.; Köksal, Mustafa; Murphy, Frank V.; Vedula, L. Sangeetha; Chou, Wayne K.W.; Cane, David E.; Christianson, David W.

2015-01-01

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with 3 Mg2+ ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed based on ~36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis. PMID:26598179

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

PubMed Central

Warshel, Arieh; Bora, Ram Prasad

2016-01-01

Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzyme’s conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions. PMID:27179464

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis.

PubMed

Warshel, Arieh; Bora, Ram Prasad

2016-05-14

Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzyme's conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions.

Copper plasmonics and catalysis: role of electron-phonon interactions in dephasing localized surface plasmons

NASA Astrophysics Data System (ADS)

Sun, Qi-C.; Ding, Yuchen; Goodman, Samuel M.; H. Funke, Hans; Nagpal, Prashant

2014-10-01

Copper metal can provide an important alternative for the development of efficient, low-cost and low-loss plasmonic nanoparticles, and selective nanocatalysts. However, poor chemical stability and lack of insight into photophysics and plasmon decay mechanisms has impeded study. Here, we use smooth conformal ALD coating on copper nanoparticles to prevent surface oxidation, and study dephasing time for localized surface plasmons on different sized copper nanoparticles. Using dephasing time as a figure of merit, we elucidate the role of electron-electron, electron-phonon, impurity, surface and grain boundary scattering on the decay of localized surface plasmon waves. Using our quantitative analysis and different temperature dependent measurements, we show that electron-phonon interactions dominate over other scattering mechanisms in dephasing plasmon waves. While interband transitions in copper metal contributes substantially to plasmon losses, tuning surface plasmon modes to infrared frequencies leads to a five-fold enhancement in the quality factor. These findings demonstrate that conformal ALD coatings can improve the chemical stability for copper nanoparticles, even at high temperatures (>300 °C) in ambient atmosphere, and nanoscaled copper is a good alternative material for many potential applications in nanophotonics, plasmonics, catalysis and nanoscale electronics.Copper metal can provide an important alternative for the development of efficient, low-cost and low-loss plasmonic nanoparticles, and selective nanocatalysts. However, poor chemical stability and lack of insight into photophysics and plasmon decay mechanisms has impeded study. Here, we use smooth conformal ALD coating on copper nanoparticles to prevent surface oxidation, and study dephasing time for localized surface plasmons on different sized copper nanoparticles. Using dephasing time as a figure of merit, we elucidate the role of electron-electron, electron-phonon, impurity, surface and grain boundary scattering on the decay of localized surface plasmon waves. Using our quantitative analysis and different temperature dependent measurements, we show that electron-phonon interactions dominate over other scattering mechanisms in dephasing plasmon waves. While interband transitions in copper metal contributes substantially to plasmon losses, tuning surface plasmon modes to infrared frequencies leads to a five-fold enhancement in the quality factor. These findings demonstrate that conformal ALD coatings can improve the chemical stability for copper nanoparticles, even at high temperatures (>300 °C) in ambient atmosphere, and nanoscaled copper is a good alternative material for many potential applications in nanophotonics, plasmonics, catalysis and nanoscale electronics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04719b

Chemical exchange in biomacromolecules: Past, present, and future

PubMed Central

Palmer, Arthur G.

2014-01-01

The perspective reviews quantitative investigations of chemical exchange phenomena in proteins and other biological macromolecules using NMR spectroscopy, particularly relaxation dispersion methods. The emphasis is on techniques and applications that quantify the populations, interconversion kinetics, and structural features of sparsely populated conformational states in equilibrium with a highly populated ground state. Applications to folding, mol ecular recognition, catalysis, and allostery by proteins and nucleic acids are highlighted. PMID:24656076

Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein*

PubMed Central

Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P.; Ke, Ailong

2012-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg2+ or Mn2+), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop. PMID:22942283

Effect of the Crystal Environment on Side-Chain Conformational Dynamics in Cyanovirin-N Investigated through Crystal and Solution Molecular Dynamics Simulations

PubMed Central

Ahlstrom, Logan S.; Vorontsov, Ivan I.; Shi, Jun; Miyashita, Osamu

2017-01-01

Side chains in protein crystal structures are essential for understanding biochemical processes such as catalysis and molecular recognition. However, crystal packing could influence side-chain conformation and dynamics, thus complicating functional interpretations of available experimental structures. Here we investigate the effect of crystal packing on side-chain conformational dynamics with crystal and solution molecular dynamics simulations using Cyanovirin-N as a model system. Side-chain ensembles for solvent-exposed residues obtained from simulation largely reflect the conformations observed in the X-ray structure. This agreement is most striking for crystal-contacting residues during crystal simulation. Given the high level of correspondence between our simulations and the X-ray data, we compare side-chain ensembles in solution and crystal simulations. We observe large decreases in conformational entropy in the crystal for several long, polar and contacting residues on the protein surface. Such cases agree well with the average loss in conformational entropy per residue upon protein folding and are accompanied by a change in side-chain conformation. This finding supports the application of surface engineering to facilitate crystallization. Our simulation-based approach demonstrated here with Cyanovirin-N establishes a framework for quantitatively comparing side-chain ensembles in solution and in the crystal across a larger set of proteins to elucidate the effect of the crystal environment on protein conformations. PMID:28107510

Effect of the Crystal Environment on Side-Chain Conformational Dynamics in Cyanovirin-N Investigated through Crystal and Solution Molecular Dynamics Simulations.

PubMed

Ahlstrom, Logan S; Vorontsov, Ivan I; Shi, Jun; Miyashita, Osamu

2017-01-01

Side chains in protein crystal structures are essential for understanding biochemical processes such as catalysis and molecular recognition. However, crystal packing could influence side-chain conformation and dynamics, thus complicating functional interpretations of available experimental structures. Here we investigate the effect of crystal packing on side-chain conformational dynamics with crystal and solution molecular dynamics simulations using Cyanovirin-N as a model system. Side-chain ensembles for solvent-exposed residues obtained from simulation largely reflect the conformations observed in the X-ray structure. This agreement is most striking for crystal-contacting residues during crystal simulation. Given the high level of correspondence between our simulations and the X-ray data, we compare side-chain ensembles in solution and crystal simulations. We observe large decreases in conformational entropy in the crystal for several long, polar and contacting residues on the protein surface. Such cases agree well with the average loss in conformational entropy per residue upon protein folding and are accompanied by a change in side-chain conformation. This finding supports the application of surface engineering to facilitate crystallization. Our simulation-based approach demonstrated here with Cyanovirin-N establishes a framework for quantitatively comparing side-chain ensembles in solution and in the crystal across a larger set of proteins to elucidate the effect of the crystal environment on protein conformations.

The conformational free-energy landscape of β-D-mannopyranose: evidence for a (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary in β-mannosidases.

PubMed

Ardèvol, Albert; Biarnés, Xevi; Planas, Antoni; Rovira, Carme

2010-11-17

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a (1)S(5) conformation, and recent experiments have proposed an unusual substrate conformational pathway ((1)S(5) → B(2,5) → (O)S(2)) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that (1)S(5) is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH.

Conformational Transition Pathways in Signaling and Enzyme Catalysis Explored by Computational Methods

NASA Astrophysics Data System (ADS)

Pachov, Dimitar V.

Biomolecules are dynamic in nature and visit a number of states while performing their biological function. However, understanding how they interconvert between functional substates is a challenging task. In this thesis, we employ enhanced computational strategies to reveal in atomistic resolution transition states and molecular mechanism along conformational pathways of the signaling protein Nitrogen Regulatory Protein C (NtrC) and the enzyme Adenylate Kinase (Adk). Targeted Molecular Dynamics (TMD) simulations and NMR experiments have previously found the active/inactive interconversion of NtrC is stabilized by non-native transient contacts. To find where along the conformational pathway they lie and probe the existence of multiple intermediates, a beyond 8mus-extensive mapping of the conformational landscape was performed by a multitude of straightforward MD simulations relaxed from the biased TMD pathway. A number of metastable states stabilized by local interactions was found to underline the conformational pathway of NtrC. Two spontaneous transitions of the last stage of the active-to-inactive conversion were identified and used in path sampling procedures to generate an ensemble of truly dynamic reactive pathways. The transition state ensemble (TSE) and mechanistic descriptors of this transition were revealed in atomic detail and verified by committor analysis. By analyzing how pressure affects the dynamics and function of two homologous Adk proteins - the P.Profundum Adk surviving at 700atm pressure in the deep sea, and the E. coli Adk that lives at ambient pressures - we indirectly obtained atomic information about the TSE of the large-amplitude rate-limiting conformational opening of the Adk lids. Guided by NMR experiments showing significantly decreased activation volumes of the piezophile compared to its mesophilic counterpart, TMD simulations revealed the formation of an extended hydrogen-bonded water network in the transition state of the piezophile that can explain the experimentally measured activation volume differences. The transition state of the conformational change was proposed to lie close to the closed state. Additionally, a number of descriptors were used to characterize the free energy landscape of the mesophile. It was found that the features of landscape are highly sensitive to the binding of different ligands, their protonation states and the presence of magnesium.

Orotidine 5'-Monophosphate Decarboxylase: Probing the Limits of the Possible for Enzyme Catalysis.

PubMed

Richard, John P; Amyes, Tina L; Reyes, Archie C

2018-04-17

The mystery associated with catalysis by what were once regarded as protein black boxes, diminished with the X-ray crystallographic determination of the three-dimensional structures of enzyme-substrate complexes. The report that several high-resolution X-ray crystal structures of orotidine 5'-monophosphate decarboxylase (OMPDC) failed to provide a consensus mechanism for enzyme-catalyzed decarboxylation of OMP to form uridine 5'-monophosphate, therefore, provoked a flurry of controversy. This controversy was fueled by the enormous 10 23 -fold rate acceleration for this enzyme, which had " jolted many biochemists' assumptions about the catalytic potential of enzymes." Our studies on the mechanism of action of OMPDC provide strong evidence that catalysis by this enzyme is not fundamentally different from less proficient catalysts, while highlighting important architectural elements that enable a peak level of performance. Many enzymes undergo substrate-induced protein conformational changes that trap their substrates in solvent occluded protein cages, but the conformational change induced by ligand binding to OMPDC is incredibly complex, as required to enable the development of 22 kcal/mol of stabilizing binding interactions with the phosphodianion and ribosyl substrate fragments of OMP. The binding energy from these fragments is utilized to activate OMPDC for catalysis of decarboxylation at the orotate fragment of OMP, through the creation of a tight, catalytically active, protein cage from the floppy, open, unliganded form of OMPDC. Such utilization of binding energy for ligand-driven conformational changes provides a general mechanism to obtain specificity in transition state binding. The rate enhancement that results from the binding of carbon acid substrates to enzymes is partly due to a reduction in the carbon acid p K a that is associated with ligand binding. The binding of UMP to OMPDC results in an unusually large >12 unit decrease in the p K a = 29 for abstraction of the C-6 substrate hydrogen, due to stabilization of an enzyme-bound vinyl carbanion, which is also an intermediate of OMPDC-catalyzed decarboxylation. The protein-ligand interactions operate to stabilize the vinyl carbanion at the enzyme active site compared to aqueous solution, rather than to stabilize the transition state for the concerted electrophilic displacement of CO 2 by H + that avoids formation of this reaction intermediate. There is evidence that OMPDC induces strain into the bound substrate. The interaction between the amide side chain of Gln-215 from the phosphodianion gripper loop and the hydroxymethylene side chain of Ser-154 from the pyrimidine umbrella of ScOMPDC position the amide side chain to interact with the phosphodianion of OMP. There are no direct stabilizing interactions between dianion gripper protein side chains Gln-215, Tyr-217, and Arg-235 and the pyrimidine ring at the decarboxylation transition state. Rather these side chains function solely to hold OMPDC in the catalytically active closed conformation. The hydrophobic side chains that line the active site of OMPDC in the region of the departing CO 2 product may function to stabilize the decarboxylation transition state by providing hydrophobic solvation of this product.

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

PubMed Central

Francis, Kevin; Sapienza, Paul J.; Lee, Andrew L.; Kohen, Amnon

2016-01-01

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general. PMID:26813442

Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

NASA Astrophysics Data System (ADS)

Pan, Xiaoliang; Schwartz, Steven

2015-03-01

It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.

Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

PubMed

Pan, Xiaoliang; Schwartz, Steven D

2015-04-30

It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

Mycobacterial nicotinate mononucleotide adenylyltransferase: Structure, mechanism, and implications for drug discovery

DOE PAGES

Rodionova, Irina A.; Zuccola, Harmon J.; Sorci, Leonardo; ...

2015-01-28

Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD ( MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect themore » structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity ( Ki ~ 25 μM) and antimycobacterial activity (MIC 80) ~ 40-80 μM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. Lastly, these findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.« less

Taking Ockham's razor to enzyme dynamics and catalysis.

PubMed

Glowacki, David R; Harvey, Jeremy N; Mulholland, Adrian J

2012-01-29

The role of protein dynamics in enzyme catalysis is a matter of intense current debate. Enzyme-catalysed reactions that involve significant quantum tunnelling can give rise to experimental kinetic isotope effects with complex temperature dependences, and it has been suggested that standard statistical rate theories, such as transition-state theory, are inadequate for their explanation. Here we introduce aspects of transition-state theory relevant to the study of enzyme reactivity, taking cues from chemical kinetics and dynamics studies of small molecules in the gas phase and in solution--where breakdowns of statistical theories have received significant attention and their origins are relatively better understood. We discuss recent theoretical approaches to understanding enzyme activity and then show how experimental observations for a number of enzymes may be reproduced using a transition-state-theory framework with physically reasonable parameters. Essential to this simple model is the inclusion of multiple conformations with different reactivity.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

PubMed

Endrizzi, James A; Beernink, Peter T

2017-11-01

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations. © 2017 The Protein Society.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis.

PubMed

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-02-09

PR65 is the two-layered (alpha-alpha solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix-helix separation along one layer ("fracturing") leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme's substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A's response to spindle tension) or arise spontaneously (e.g., in PP2A's interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer's-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

PubMed Central

Grinthal, Alison; Adamovic, Ivana; Weiner, Beth; Karplus, Martin; Kleckner, Nancy

2010-01-01

PR65 is the two-layered (α-α solenoid) HEAT-repeat (Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, PI3 kinase target of rapamycin 1) scaffold of protein phosphatase PP2A. Molecular dynamics simulations predict that, at forces expected in living systems, PR65 undergoes (visco-)elastic deformations in response to pulling/pushing on its ends. At lower forces, smooth global flexural and torsional changes occur via even redistribution of stress along the hydrophobic core of the molecule. At intermediate forces, helix–helix separation along one layer (“fracturing”) leads to global relaxation plus loss of contact in the other layer to unstack the affected units. Fracture sites are determined by unusual sequences in contiguous interhelix turns. Normal mode analysis of the heterotrimeric PP2A enzyme reveals that its ambient conformational fluctuations are dominated by elastic deformations of PR65, which introduce a mechanical linkage between the separately bound regulatory and catalytic subunits. PR65-dominated fluctuations of PP2A have the effect of opening and closing the enzyme’s substrate binding/catalysis interface, as well as altering the positions of certain catalytic residues. These results suggest that substrate binding/catalysis are sensitive to mechanical force. Force could be imposed from the outside (e.g., in PP2A’s response to spindle tension) or arise spontaneously (e.g., in PP2A’s interaction with unstructured proteins such as Tau, a microtubule-associated Alzheimer’s-implicated protein). The presented example supports the view that conformation and function of protein complexes can be modulated by mechanical energy inputs, as well as by chemical energy inputs from ligand binding. Given that helical-repeat proteins are involved in many cellular processes, the findings also encourage the view that mechanical forces may be of widespread importance. PMID:20133745

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

NASA Astrophysics Data System (ADS)

Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; de Proft, Frank; Huang, Jingjing; van Breusegem, Frank; Messens, Joris

2017-02-01

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

PubMed Central

Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; De Proft, Frank; Huang, Jingjing; Van Breusegem, Frank; Messens, Joris

2017-01-01

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. PMID:28195196

Reversible Regulation of Catalytic Activity of Gold Nanoparticles with DNA Nanomachines

NASA Astrophysics Data System (ADS)

Zhou, Peipei; Jia, Sisi; Pan, Dun; Wang, Lihua; Gao, Jimin; Lu, Jianxin; Shi, Jiye; Tang, Zisheng; Liu, Huajie

2015-09-01

Reversible catalysis regulation has gained much attention and traditional strategies utilized reversible ligand coordination for switching catalyst’s conformations. However, it remains challenging to regulate the catalytic activity of metal nanoparticle-based catalysts. Herein, we report a new DNA nanomachine-driven reversible nano-shield strategy for circumventing this problem. The basic idea is based on the fact that the conformational change of surface-attached DNA nanomachines will cause the variation of the exposed surface active area on metal nanoparticles. As a proof-of-concept study, we immobilized G-rich DNA strands on gold nanoparticles (AuNPs) which have glucose oxidase (GOx) like activity. Through the reversible conformational change of the G-rich DNA between a flexible single-stranded form and a compact G-quadruplex form, the catalytic activity of AuNPs has been regulated reversibly for several cycles. This strategy is reliable and robust, which demonstrated the possibility of reversibly adjusting catalytic activity with external surface coverage switching, rather than coordination interactions.

The N253F mutant structure of trehalose synthase from Deinococcus radiodurans reveals an open active-site topology.

PubMed

Chow, Sih Yao; Wang, Yung Lin; Hsieh, Yu Chiao; Lee, Guan Chiun; Liaw, Shwu Huey

2017-11-01

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes.

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

PubMed

Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Detection techniques for tenuous planetary atmospheres

NASA Technical Reports Server (NTRS)

Hoenig, S. A.

1972-01-01

The research for the development of new types of detectors for analysis of planetary atmospheres is summarized. Topics discussed include: corona discharge humidity detector, surface catalysis and exo-electron emission, and analysis of soil samples by means of exo-electron emission. A report on the exo-electron emission during heterogeneous catalysis is included.

Hydrogen bonding in the mechanism of GDP-mannose mannosyl hydrolase

NASA Astrophysics Data System (ADS)

Mildvan, A. S.; Xia, Z.; Azurmendi, H. F.; Legler, P. M.; Balfour, M. R.; Lairson, L. L.; Withers, S. G.; Gabelli, S. B.; Bianchet, M. A.; Amzel, L. M.

2006-06-01

GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α- D-sugars to GDP and β- D-sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 Å X-ray structure of the GDPMH-Mg 2+-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg 2+. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-NδH⋯Tyr-127-OH⋯Pro-120(C dbnd6 O). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both kcat and Km. The GDP leaving group is activated by the essential Mg 2+ which contributes at least 10 5-fold to kcat, and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to kcat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on kcat, indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 10 2.1- to 10 2.6-fold to kcat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2-OH of the mannosyl group, and His-88, which contributes 10 2.3-fold to kcat, is positioned to accept a hydrogen bond from the C3-OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-α- D-mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower kcat and a 2.5-fold greater Km than does GDP-α- D-mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg 2+, Asp-22, His-124, and His-88 is ≥10 19, which exceeds the 10 12-fold rate acceleration produced by GDPMH by a factor ≥10 7. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.

A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis.

PubMed

Huang, Kai-Fa; Wang, Yu-Ruei; Chang, En-Cheng; Chou, Tsung-Lin; Wang, Andrew H-J

2008-04-01

QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

PubMed Central

2009-01-01

Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

Origin of a folded repeat protein from an intrinsically disordered ancestor

PubMed Central

Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

2016-01-01

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

Watching Individual Enzymes at Work

NASA Astrophysics Data System (ADS)

Blank, Kerstin; Rocha, Susana; De Cremer, Gert; Roeffaers, Maarten B. J.; Uji-i, Hiroshi; Hofkens, Johan

Single-molecule fluorescence experiments are a powerful tool to analyze reaction mechanisms of enzymes. Because of their unique potential to detect heterogeneities in space and time, they have provided unprecedented insights into the nature and mechanisms of conformational changes related to the catalytic reaction. The most important finding from experiments with single enzymes is the generally observed phenomenon that the catalytic rate constants fluctuate over time (dynamic disorder). These fluctuations originate from conformational changes occurring on time scales, which are similar to or slower than that of the catalytic reaction. Here, we summarize experiments with enzymes that show dynamic disorder and introduce new experimental strategies showing how single-molecule fluorescence experiments can be applied to address other open questions in medical and industrial enzymology, such as enzyme inactivation processes, reactant transfer in cascade reactions, and the mechanisms of interfacial catalysis.

Hotspots for allosteric regulation on protein surfaces

PubMed Central

Reynolds, Kimberly A.; McLaughlin, Richard N.; Ranganathan, Rama

2012-01-01

Recent work indicates a general architecture for proteins in which sparse networks of physically contiguous and co-evolving amino acids underlie basic aspects of structure and function. These networks, termed sectors, are spatially organized such that active sites are linked to many surface sites distributed throughout the structure. Using the metabolic enzyme dihydrofolate reductase as a model system, we show that (1) the sector is strongly correlated to a network of residues undergoing millisecond conformational fluctuations associated with enzyme catalysis and (2) sector-connected surface sites are statistically preferred locations for the emergence of allosteric control in vivo. Thus, sectors represent an evolutionarily conserved “wiring” mechanism that can enable perturbations at specific surface positions to rapidly initiate conformational control over protein function. These findings suggest that sectors enable the evolution of intermolecular communication and regulation. PMID:22196731

Oxidative and photoxidative polymerization of humic suprastructures by heterogeneous biomimetic catalysis.

PubMed

Nuzzo, Assunta; Piccolo, Alessandro

2013-05-13

The meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of manganese(III) chloride [Mn-(TDCPPS)Cl] biomimetic catalyst immobilized on spacer-functionalized kaolinite clay mineral was employed in the oxidative coupling reaction of a dissolved humic acid (HA) suprastructure with either chemical (H2O2) or UV-light oxidation. The changes in molecular size of humic matter subjected to catalyzed oxidative reaction were followed by high-performance size exclusion chromatography (HPSEC) with UV-vis and refractive index (RI) detectors in series, and by thermogravimetric (TGA) analysis. Both the enhanced molecular size shown by differences between HPSEC chromatograms of humic reaction mixtures at either pH 6 or 3.5 and the increase of thermogravimetric stability suggest that the heterogeneous biomimetic catalysis promoted the stabilization of humic conformations by new intermolecular covalent bonds during oxidative coupling. The similarity between chemical and light-induced oxidation results suggests potential multiple applications of the kaolinite-supported heterogeneous catalyst in controlling the reactivity of natural organic matter within biogeochemical cycles and environmental reactions.

Measuring Conformational Dynamics of Single Biomolecules Using Nanoscale Electronic Devices

NASA Astrophysics Data System (ADS)

Akhterov, Maxim V.; Choi, Yongki; Sims, Patrick C.; Olsen, Tivoli J.; Gul, O. Tolga; Corso, Brad L.; Weiss, Gregory A.; Collins, Philip G.

2014-03-01

Molecular motion can be a rate-limiting step of enzyme catalysis, but motions are typically too quick to resolve with fluorescent single molecule techniques. Recently, we demonstrated a label-free technique that replaced fluorophores with nano-electronic circuits to monitor protein motions. The solid-state electronic technique used single-walled carbon nanotube (SWNT) transistors to monitor conformational motions of a single molecule of T4 lysozyme while processing its substrate, peptidoglycan. As lysozyme catalyzes the hydrolysis of glycosidic bonds, two protein domains undergo 8 Å hinge bending motion that generates an electronic signal in the SWNT transistor. We describe improvements to the system that have extended our temporal resolution to 2 μs . Electronic recordings at this level of detail directly resolve not just transitions between open and closed conformations but also the durations for those transition events. Statistical analysis of many events determines transition timescales characteristic of enzyme activity and shows a high degree of variability within nominally identical chemical events. The high resolution technique can be readily applied to other complex biomolecules to gain insights into their kinetic parameters and catalytic function.

Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

PubMed Central

Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

2015-01-01

Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

PubMed Central

Doshi, Urmi; Holliday, Michael J.; Eisenmesser, Elan Z.; Hamelberg, Donald

2016-01-01

Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue–residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general. PMID:27071107

H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

PubMed Central

Bettridge, John; Na, Chan Hyun; Desiderio, Stephen

2017-01-01

V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin. PMID:28174273

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Short peptides self-assemble to produce catalytic amyloids

NASA Astrophysics Data System (ADS)

Rufo, Caroline M.; Moroz, Yurii S.; Moroz, Olesia V.; Stöhr, Jan; Smith, Tyler A.; Hu, Xiaozhen; Degrado, William F.; Korendovych, Ivan V.

2014-04-01

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of seven-residue peptides that act as Zn2+-dependent esterases. Zn2+ helps stabilize the fibril formation, while also acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyse their own formation, but they can also catalyse chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and non-biological metal ions.

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function

PubMed Central

Lisi, George P.; Loria, J. Patrick

2015-01-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. PMID:26952190

Programmable Self-assembly of Hydrocarbon-capped Nanoparticles: Role of Chain Conformations

NASA Astrophysics Data System (ADS)

Waltmann, Curt; Horst, Nathan; Travesset, Alex

Nanoparticle superlattices (NPS), i.e. crystalline arrangements of nanoparticles, are materials with fascinating structures, which in many cases are not possible to attain from simple atoms or molecules. They also span a wide range of possible applications such as metamaterials, new energy sources, catalysis, and many others. In this talk, we present a theoretical and computational description of the self-assembly of nanoparticles with hydrocarbons as capping ligands. Usually, these systems have been described with hard sphere packing models. In this talk, we show that the conformations of the hydrocarbon chains play a fundamental role in determining the equilibrium phases, including and especially in binary systems. The work of CW was supported by a DOE-SULI internship from May-December 2016, and by NSF, DMR-CMMT 1606336 CDS&E: Design Principles for Ordering Nanoparticles into Super-crystals after January 1st.

Isolation and characterisation of an unexpected byproduct in the regioselective butane diacetal protection of α-methyl galactopyranoside.

PubMed

Fontenelle, Clément Q; Kuppala, Ramakrishna; Light, Mark; Linclau, Bruno

2018-01-02

The regioselective protection of both methyl galactopyranoside anomers at the 2 and 3-positions as the butane diacetal (BDA) is well known. Here we describe the formation of an unexpected byproduct, which mainly occurs when α-methyl galactopyranoside is reacted with 2,3-butanedione under BF 3 •OEt 2 catalysis. The structure of the byproduct, which did not arise from anomerisation to the β-anomer or from BDA formation at the galactopyranoside 3,4-positions, was elucidated by NMR and X-ray crystallographic analysis, and proved to be the expected BDA protected galactopyranoside, but in which the stereochemistry of both its BDA acetal centres are inverted. Interestingly, the conformation of the resulting six-membered BDA ring was distorted to a skew boat conformation in order to maintain anomeric stabilisation. Copyright © 2017. Published by Elsevier Ltd.

An allosteric photoredox catalyst inspired by photosynthetic machinery

PubMed Central

Lifschitz, Alejo M.; Young, Ryan M.; Mendez-Arroyo, Jose; Stern, Charlotte L.; McGuirk, C. Michael; Wasielewski, Michael R.; Mirkin, Chad A.

2015-01-01

Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switching using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Thus, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices. PMID:25817586

Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

PubMed Central

2015-01-01

Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context of spatially distinct differences in enthalpic barriers for protein conformational sampling that may be related to catalysis. PMID:24892947

Replacement of Ser108 in Plasmodium falciparum enolase results in weak Mg(II) binding: role of a parasite-specific pentapeptide insert in stabilizing the active conformation of the enzyme.

PubMed

Dutta, Sneha; Mukherjee, Debanjan; Jarori, Gotam K

2015-06-01

A distinct structural feature of Plasmodium falciparum enolase (Pfeno) is the presence of a five amino acid insert -104EWGWS108- that is not found in host enolases. Its conservation among apicomplexan enolases has raised the possibility of its involvement in some important physiological function(s). Deletion of this sequence is known to lower k(cat)/K(m), increase K(a) for Mg(II) and convert dimer into monomers (Vora HK, Shaik FR, Pal-Bhowmick I, Mout R & Jarori GK (2009) Arch Biochem Biophys 485, 128-138). These authors also raised the possibility of the formation of an H-bond between Ser108 and Leu49 that could stabilize the apo-Pfeno in an active closed conformation that has high affinity for Mg(II). Here, we examined the effect of replacement of Ser108 with Gly/Ala/Thr on enzyme activity, Mg(II) binding affinity, conformational states and oligomeric structure and compared it with native recombinant Pfeno. The results obtained support the view that Ser108 is likely to be involved in the formation of certain crucial H-bonds with Leu49. The presence of these interactions can stabilize apo-Pfeno in an active closed conformation similar to that of Mg(II) bound yeast enolase. As predicted, S108G/A-Pfeno variants (where Ser108-Leu49 H-bonds are likely to be disrupted) were found to exist in an open conformation and had low affinity for Mg(II). They also required Mg(II) induced conformational changes to acquire the active closed conformational state essential for catalysis. The possible physiological relevance of apo-Pfeno being in such an active state is discussed. © 2015 FEBS.

Substrate Binding Drives Active-Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

PubMed

Weissbach, Sophie; Flügge, Friedemann; Peters, Thomas

2018-05-04

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular Library Synthesis Using Complex Substrates: Expanding the Framework of Triterpenoids

PubMed Central

Ignatenko, Vasily A.; Han, Yong; Tochtrop, Gregory P.

2013-01-01

The remodelling of a natural product core framework by means of diversity-oriented synthesis (DOS) is a valuable approach to access diverse/biologically relevant chemical space and to overcome the limitations of combinatorial-type compounds. Here we provide proof of principle and a thorough conformational analysis for a general strategy whereby the inherent complexity of a starting material is used to define the regio- and stereochemical outcomes of reactions in chemical library construction. This is in contrast to the traditional DOS logic employing reaction development and catalysis to drive library diversity PMID:23245400

The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis

NASA Astrophysics Data System (ADS)

Jiang, Shuiqin; Zhang, Lujia; Cui, Dongbin; Yao, Zhiqiang; Gao, Bei; Lin, Jinping; Wei, Dongzhi

2016-10-01

The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.

Halogen bonding in solution: thermodynamics and applications.

PubMed

Beale, Thomas M; Chudzinski, Michael G; Sarwar, Mohammed G; Taylor, Mark S

2013-02-21

Halogen bonds are noncovalent interactions in which covalently bound halogens act as electrophilic species. The utility of halogen bonding for controlling self-assembly in the solid state is evident from a broad spectrum of applications in crystal engineering and materials science. Until recently, it has been less clear whether, and to what extent, halogen bonding could be employed to influence conformation, binding or reactivity in the solution phase. This tutorial review summarizes and interprets solution-phase thermodynamic data for halogen bonding interactions obtained over the past six decades and highlights emerging applications in molecular recognition, medicinal chemistry and catalysis.

Designer Shape Anisotropy on Transition-Metal-Dichalcogenide Nanosheets.

PubMed

Martella, Christian; Mennucci, Carlo; Lamperti, Alessio; Cappelluti, Emmanuele; de Mongeot, Francesco Buatier; Molle, Alessandro

2018-03-01

MoS 2 and generally speaking, the wide family of transition-metal dichalcogenides represents a solid nanotechnology platform on which to engineer a wealth of new and outperforming applications involving 2D materials. An even richer flexibility can be gained by extrinsically inducing an in-plane shape anisotropy of the nanosheets. Here, the synthesis of anisotropic MoS 2 nanosheets is proposed as a prototypical example in this respect starting from a highly conformal chemical vapor deposition on prepatterend substrates and aiming at the more general purpose of tailoring anisotropy of 2D nanosheets by design. This is envisioned to be a suitable configuration for strain engineering as far as strain can be spatially redistributed in morphologically different regions. With a similar approach, both the optical and electronic properties of the 2D transition-metal dichalcogenides can be tailored over macroscopic sample areas in a self-organized fashion, thus paving the way for new applications in the field of optical metasurfaces, light harvesting, and catalysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of oxidation on protein therapeutics: Conformational dynamics of intact and oxidized acid-β-glucocerebrosidase at near-physiological pH

PubMed Central

Bobst, Cedric E; Thomas, John J; Salinas, Paul A; Savickas, Philip; Kaltashov, Igor A

2010-01-01

The solution dynamics of an enzyme acid-β-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain. Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity. Forced oxidation of GCase that resulted in a 40–60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site. These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity. Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability. Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking. PMID:20945356

Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

PubMed

Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine; André, Bruno

2014-12-01

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Role of dynamics in enzyme catalysis: substantial versus semantic controversies.

PubMed

Kohen, Amnon

2015-02-17

CONSPECTUS: The role of the enzyme's dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term "protein dynamics" is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is "stored" in a specific protein mode and "used" for catalysis before it can dissipate to its environment (i.e., "nonstatistical dynamics"). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual "induced-fit" shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction's transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions.

Role of Dynamics in Enzyme Catalysis: Substantial versus Semantic Controversies

PubMed Central

2015-01-01

Conspectus The role of the enzyme’s dynamic motions in catalysis is at the center of heated contemporary debates among both theoreticians and experimentalists. Resolving these apparent disputes is of both intellectual and practical importance: incorporation of enzyme dynamics could be critical for any calculation of enzymatic function and may have profound implications for structure-based drug design and the design of biomimetic catalysts. Analysis of the literature suggests that while part of the dispute may reflect substantial differences between theoretical approaches, much of the debate is semantic. For example, the term “protein dynamics” is often used by some researchers when addressing motions that are in thermal equilibrium with their environment, while other researchers only use this term for nonequilibrium events. The last cases are those in which thermal energy is “stored” in a specific protein mode and “used” for catalysis before it can dissipate to its environment (i.e., “nonstatistical dynamics”). This terminology issue aside, a debate has arisen among theoreticians around the roles of nonstatistical vs statistical dynamics in catalysis. However, the author knows of no experimental findings available today that examined this question in enzyme catalyzed reactions. Another source of perhaps nonsubstantial argument might stem from the varying time scales of enzymatic motions, which range from seconds to femtoseconds. Motions at different time scales play different roles in the many events along the catalytic cascade (reactant binding, reprotonation of reactants, structural rearrangement toward the transition state, product release, etc.). In several cases, when various experimental tools have been used to probe catalytic events at differing time scales, illusory contradictions seem to have emerged. In this Account, recent attempts to sort the merits of those questions are discussed along with possible future directions. A possible summary of current studies could be that enzyme, substrate, and solvent dynamics contribute to enzyme catalyzed reactions in several ways: first via mutual “induced-fit” shifting of their conformational ensemble upon binding; then via thermal search of the conformational space toward the reaction’s transition-state (TS) and the rare event of the barrier crossing toward products, which is likely to be on faster time scales then the first and following events; and finally via the dynamics associated with products release, which are rate-limiting for many enzymatic reactions. From a chemical perspective, close to the TS, enzymatic systems seem to stiffen, restricting motions orthogonal to the chemical coordinate and enabling dynamics along the reaction coordinate to occur selectively. Studies of how enzymes evolved to support those efficient dynamics at various time scales are still in their infancy, and further experiments and calculations are needed to reveal these phenomena in both enzymes and uncatalyzed reactions. PMID:25539442

Identification of geometrical isomers and comparison of different isomeric samples of astaxanthin.

PubMed

Qiu, Dan; Wu, Yue-Chan; Zhu, Wen-Li; Yin, Hong; Yi, Long-Tao

2012-09-01

A high-performance liquid chromatographic (HPLC) analysis system for isomeric astaxanthin was developed. The separation system consisted of a C(30) column and an elution system of methanol/MTBE/water/dichloromethane (77:13:8:2, v/v/v/v). Using the combination of HPLC diode array detector and HPLC atmospheric pressure chemical ionization mass spectrometry, 11 geometrical isomers and 4 epoxides of astaxanthin were successfully identified. Referred to crystal, only isomerization with different degrees was found for solvent dissolving and iodine catalysis, while melting of astaxanthin caused isomerization, slight oxidation, and more noticeable polymerization confirmed by gel permeation chromatography. Chemical changes in isomeric samples all caused a decrease in UV content. The vibrational spectra (infrared and Raman) showed that epoxide was the only new functional group generated for melting. Changes of several key bands and formations of new bands were found in iodine catalysis and melting samples because of isomerization. Practical Application:  Eleven geometrical isomers and 4 epoxides, which were normally generated for solvent dissolving, iodine catalysis, and melting of astaxanthin, have been identified by C(30) -HPLC-MS technology. Furthermore, different samples were measured by gel permeation chromatography, UV, infrared, and Raman, based on the analysis of messages, the effect of each processing was well understood. © 2012 Institute of Food Technologists®

How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

PubMed Central

Zhang, Ji-Long; Zheng, Qing-Chuan; Li, Zheng-Qiang; Zhang, Hong-Xing

2013-01-01

The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism. PMID:23308285

How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

PubMed

Wu, Wen-Jin; Su, Mei-I; Wu, Jian-Li; Kumar, Sandeep; Lim, Liang-Hin; Wang, Chun-Wei Eric; Nelissen, Frank H T; Chen, Ming-Chuan Chad; Doreleijers, Jurgen F; Wijmenga, Sybren S; Tsai, Ming-Daw

2014-04-02

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plapp, Bryce V.; Savarimuthu, Baskar Raj; Ferraro, Daniel J.

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentatemore » chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ~1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.« less

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

PubMed Central

2017-01-01

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5′-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2′-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water. PMID:28640600

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

PubMed

Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

2004-03-09

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Low Cost Solar Array Project: Composition Measurements by Analytical Photon Catalysis

NASA Technical Reports Server (NTRS)

Sutton, D. G.; Galvan, L.; Melzer, J.; Heidner, R. F., III

1979-01-01

The applicability of the photon catalysis technique for effecting composition analysis of silicon samples was assessed. Third quarter activities were devoted to the study of impurities in silicon matrices. The evaporation process was shown to be congruent; thus, the spectral analysis of the vapor yields the composition of the bulk sample. Qualitative analysis of metal impurities in silicon was demonstrated e part per million level. Only one atomic spectral interference was noted; however, it is imperative to maintain a leak tight system due to chemical and spectral interferences caused by the presence of even minute amounts of oxygen in the active nitrogen afterglow.

Structural Basis of the Induced-Fit Mechanism of 1,4-Dihydroxy-2-Naphthoyl Coenzyme A Synthase from the Crotonase Fold Superfamily

PubMed Central

Li, Jie; Li, Yan; Jiang, Ming; Zhou, Jiahai; Guo, Zhihong

2013-01-01

1, 4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase fold enzyme with an implicated role of conformational changes in catalysis. We have identified these conformational changes by determining the structures of its Escherichia coli and Synechocystis sp. PCC6803 orthologues in complex with a product analog. The structural changes include the folding of an active-site loop into a β-hairpin and significant reorientation of a helix at the carboxy terminus. Interestingly, a new interface is formed between the ordered loop and the reoriented helix, both of which also form additional interactions with the coenzyme A moiety of the ligand. Site-directed mutation of the amino acid residues involved in these ligand-induced interactions significantly diminishes the enzyme activity. These results suggest a catalytically essential induced-fit that is likely initiated by the enzyme-ligand interactions at the active site. PMID:23658663

X-ray Crystal Structures Elucidate the Nucleotidyl Transfer Reaction of Transcript Initiation Using Two Nucleotides

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Gleghorn; E Davydova; R Basu

2011-12-31

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groupsmore » of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.« less

ANI-1, A data set of 20 million calculated off-equilibrium conformations for organic molecules

NASA Astrophysics Data System (ADS)

Smith, Justin S.; Isayev, Olexandr; Roitberg, Adrian E.

2017-12-01

One of the grand challenges in modern theoretical chemistry is designing and implementing approximations that expedite ab initio methods without loss of accuracy. Machine learning (ML) methods are emerging as a powerful approach to constructing various forms of transferable atomistic potentials. They have been successfully applied in a variety of applications in chemistry, biology, catalysis, and solid-state physics. However, these models are heavily dependent on the quality and quantity of data used in their fitting. Fitting highly flexible ML potentials, such as neural networks, comes at a cost: a vast amount of reference data is required to properly train these models. We address this need by providing access to a large computational DFT database, which consists of more than 20 M off equilibrium conformations for 57,462 small organic molecules. We believe it will become a new standard benchmark for comparison of current and future methods in the ML potential community.

A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation

PubMed Central

Schellenberg, Matthew J.; Wu, Tao; Ritchie, Dustin B.; Fica, Sebastian; Staley, Jonathan P.; Atta, Karim A.; LaPointe, Paul; MacMillan, Andrew M.

2013-01-01

SUMMARY Splicing of pre-mRNAs in eukaryotes is catalyzed by the spliceosome a large RNA–protein metalloenzyme. The catalytic center of the spliceosome involves a structure comprised of the U2 and U6 snRNAs and includes a metal bound by U6 snRNA. The precise architecture of the splicesome active site however, including the question of whether it includes protein components, remains unresolved. A wealth of evidence places the protein PRP8 at the heart of the spliceosome through assembly and catalysis. Here we provide evidence that the RNase H domain of PRP8 undergoes a conformational switch between the two steps of splicing rationalizing yeast prp8 alleles promoting either the first or second step. We also show that this switch unmasks a metal-binding site involved in the second step. Together these data establish that PRP8 is a metalloprotein that promotes exon ligation within the spliceosome. PMID:23686287

Using NMR spectroscopy to elucidate the role of molecular motions in enzyme function.

PubMed

Lisi, George P; Loria, J Patrick

2016-02-01

Conformational motions play an essential role in enzyme function, often facilitating the formation of enzyme-substrate complexes and/or product release. Although considerable debate remains regarding the role of molecular motions in the conversion of enzymatic substrates to products, numerous examples have found motions to be crucial for optimization of enzyme scaffolds, effective substrate binding, and product dissociation. Conformational fluctuations are often rate-limiting to enzyme catalysis, primarily through product release, with the chemical reaction occurring much more quickly. As a result, the direct involvement of motions at various stages along the enzyme reaction coordinate remains largely unknown and untested. In the following review, we describe the use of solution NMR techniques designed to probe various timescales of molecular motions and detail examples in which motions play a role in propagating catalytic effects from the active site and directly participate in essential aspects of enzyme function. Copyright © 2015 Elsevier B.V. All rights reserved.

Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

PubMed Central

Fuller, James R; Rice, Phoebe A

2017-01-01

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal. DOI: http://dx.doi.org/10.7554/eLife.21777.001 PMID:28177285

An allosteric photoredox catalyst inspired by photosynthetic machinery

DOE PAGES

Lifschitz, Alejo M.; Young, Ryan M.; Mendez-Arroyo, Jose; ...

2015-03-30

Biological photosynthetic machinery allosterically regulate light harvesting via conformational and electronic changes at the antenna protein complexes as a response to specific chemical inputs. Fundamental limitations in current approaches to regulating inorganic light-harvesting mimics prevent their use in catalysis. Here we show that a light-harvesting antenna/reaction centre mimic can be regulated by utilizing a coordination framework incorporating antenna hemilabile ligands and assembled via a high-yielding, modular approach. As in nature, allosteric regulation is afforded by coupling the conformational changes to the disruptions in the electrochemical landscape of the framework upon recognition of specific coordinating analytes. The hemilabile ligands enable switchingmore » using remarkably mild and redox-inactive inputs, allowing one to regulate the photoredox catalytic activity of the photosynthetic mimic reversibly and in situ. Furthermore, we demonstrate that bioinspired regulatory mechanisms can be applied to inorganic light-harvesting arrays displaying switchable catalytic properties and with potential uses in solar energy conversion and photonic devices.« less

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

PubMed Central

Watanabe, Rikiya; Hayashi, Kumiko; Ueno, Hiroshi; Noji, Hiroyuki

2013-01-01

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F1-ATPase (F1) by attaching probes with different viscous drag coefficients at the rotary shaft of F1. Individual rotation pauses of F1 between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F1 rate enhancement. PMID:24268150

Statistical mechanical model of gas adsorption in porous crystals with dynamic moieties

PubMed Central

Braun, Efrem; Carraro, Carlo; Smit, Berend

2017-01-01

Some nanoporous, crystalline materials possess dynamic constituents, for example, rotatable moieties. These moieties can undergo a conformation change in response to the adsorption of guest molecules, which qualitatively impacts adsorption behavior. We pose and solve a statistical mechanical model of gas adsorption in a porous crystal whose cages share a common ligand that can adopt two distinct rotational conformations. Guest molecules incentivize the ligands to adopt a different rotational configuration than maintained in the empty host. Our model captures inflections, steps, and hysteresis that can arise in the adsorption isotherm as a signature of the rotating ligands. The insights disclosed by our simple model contribute a more intimate understanding of the response and consequence of rotating ligands integrated into porous materials to harness them for gas storage and separations, chemical sensing, drug delivery, catalysis, and nanoscale devices. Particularly, our model reveals design strategies to exploit these moving constituents and engineer improved adsorbents with intrinsic thermal management for pressure-swing adsorption processes. PMID:28049851

Statistical mechanical model of gas adsorption in porous crystals with dynamic moieties.

PubMed

Simon, Cory M; Braun, Efrem; Carraro, Carlo; Smit, Berend

2017-01-17

Some nanoporous, crystalline materials possess dynamic constituents, for example, rotatable moieties. These moieties can undergo a conformation change in response to the adsorption of guest molecules, which qualitatively impacts adsorption behavior. We pose and solve a statistical mechanical model of gas adsorption in a porous crystal whose cages share a common ligand that can adopt two distinct rotational conformations. Guest molecules incentivize the ligands to adopt a different rotational configuration than maintained in the empty host. Our model captures inflections, steps, and hysteresis that can arise in the adsorption isotherm as a signature of the rotating ligands. The insights disclosed by our simple model contribute a more intimate understanding of the response and consequence of rotating ligands integrated into porous materials to harness them for gas storage and separations, chemical sensing, drug delivery, catalysis, and nanoscale devices. Particularly, our model reveals design strategies to exploit these moving constituents and engineer improved adsorbents with intrinsic thermal management for pressure-swing adsorption processes.

Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices in Living Cells.

PubMed

Chen, Feng; Bai, Min; Cao, Ke; Zhao, Yue; Cao, Xiaowen; Wei, Jing; Wu, Na; Li, Jiang; Wang, Lihua; Fan, Chunhai; Zhao, Yongxi

2017-12-26

Molecular nanodevices are computational assemblers that switch defined states upon external stimulation. However, interfacing artificial nanodevices with natural molecular machineries in living cells remains a great challenge. Here, we delineate a generic method for programming assembly of enzyme-initiated DNAzyme nanodevices (DzNanos). Two programs including split assembly of two partzymes and toehold exchange displacement assembly of one intact DNAzyme initiated by telomerase are computed. The intact one obtains higher assembly yield and catalytic performance ascribed to proper conformation folding and active misplaced assembly. By employing MnO 2 nanosheets as both DNA carriers and source of Mn 2+ as DNAzyme cofactor, we find that this DzNano is well assembled via a series of conformational states in living cells and operates autonomously with sustained cleavage activity. Other enzymes can also induce corresponding DzNano assembly with defined programming modules. These DzNanos not only can monitor enzyme catalysis in situ but also will enable the implementation of cellular stages, behaviors, and pathways for basic science, diagnostic, and therapeutic applications as genetic circuits.

X-ray absorption spectroscopy to watch catalysis by metalloenzymes: status and perspectives discussed for the water-splitting manganese complex of photosynthesis.

PubMed

Dau, Holger; Haumann, Michael

2003-01-01

Understanding structure-function relations is one of the main interests in the molecular biosciences. X-ray absorption spectroscopy of biological samples (BioXAS) has gained the status of a useful tool for characterization of the structure of protein-bound metal centers with respect to the electronic structure (oxidation states, orbital occupancies) and atomic structure (arrangement of ligand atoms). Owing to progress in the performance characteristics of synchrotron radiation sources and of experimental stations dedicated to the study of (ultra-dilute) biological samples, it is now possible to carry out new types of BioXAS experiments, which have been impracticable in the past. Of particular interest are approaches to follow biological catalysis at metal sites by characterization of functionally relevant structural changes. In this Article, the first steps towards the use of BioXAS to 'watch' biological catalysis are reviewed for the water-splitting reactions occurring at the manganese complex of photosynthesis. The following aspects are considered: the role of BioXAS in life sciences; methodological aspects of BioXAS; catalysis at the Mn complex of photosynthesis; combination of EXAFS and crystallographic information; the freeze-quench technique to capture semi-stable states; time-resolved BioXAS using a freeze-quench approach; room-temperature experiments and 'real-time' BioXAS; tasks and perspectives.

Toward the accurate first-principles prediction of ionization equilibria in proteins.

PubMed

Khandogin, Jana; Brooks, Charles L

2006-08-08

The calculation of pK(a) values for ionizable sites in proteins has been traditionally based on numerical solutions of the Poisson-Boltzmann equation carried out using a high-resolution protein structure. In this paper, we present a method based on continuous constant pH molecular dynamics (CPHMD) simulations, which allows the first-principles description of protein ionization equilibria. Our method utilizes an improved generalized Born implicit solvent model with an approximate Debye-Hückel screening function to account for salt effects and the replica-exchange (REX) protocol for enhanced conformational and protonation state sampling. The accuracy and robustness of the present method are demonstrated by 1 ns REX-CPHMD titration simulations of 10 proteins, which exhibit anomalously large pK(a) shifts for the carboxylate and histidine side chains. The experimental pK(a) values of these proteins are reliably reproduced with a root-mean-square error ranging from 0.6 unit for proteins containing few buried ionizable side chains to 1.0 unit or slightly higher for proteins containing ionizable side chains deeply buried in the core and experiencing strong charge-charge interactions. This unprecedented level of agreement with experimental benchmarks for the de novo calculation of pK(a) values suggests that the CPHMD method is maturing into a practical tool for the quantitative prediction of protein ionization equilibria, and this, in turn, opens a door to atomistic simulations of a wide variety of pH-coupled conformational phenomena in biological macromolecules such as protein folding or misfolding, aggregation, ligand binding, membrane interaction, and catalysis.

Gold coated metal nanostructures grown by glancing angle deposition and pulsed electroplating

NASA Astrophysics Data System (ADS)

Grüner, Christoph; Reeck, Pascal; Jacobs, Paul-Philipp; Liedtke, Susann; Lotnyk, Andriy; Rauschenbach, Bernd

2018-05-01

Nickel based nanostructures are grown by glancing angle deposition (GLAD) on flat and pre-patterned substrates. These fabricated porous thin films were subsequently coated by pulsed electroplating with gold. The morphology and conformity of the gold coating were investigated by scanning electron microscopy and X-ray diffraction. Controlled growth of closed gold layers on the nanostructures could be achieved, while the open-pore structure of the nanosculptured thin films was preserved. Such gold coated nanostructures are a candidate for optical sensing and catalysis applications. The demonstrated method can be applied for numerous material combinations, allowing to provide GLAD thin films with new surface properties.

At the Dawn of the 21st Century: Is Dynamics the Missing Link for Understanding Enzyme Catalysis?

PubMed Central

Kamerlin, Shina C. L.; Warshel, Arieh

2009-01-01

Enzymes play a key role in almost all biological processes, accelerating a variety of metabolic reactions as well as controlling energy transduction, transcription and translation of genetic information, and signaling. They possess the remarkable capacity to accelerate reactions by many orders of magnitude compared to their uncatalyzed counterparts, making feasible crucial processes that would otherwise not occur on biologically relevant timescales. Thus, there is broad interest in understanding the catalytic power of enzymes on a molecular level. Several proposals have been put forward to try to explain this phenomenon, and one that has rapidly gained momentum in recent years is the idea that enzyme dynamics somehow contributes to catalysis. This review examines the dynamical proposal in a critical way considering basically all reasonable definitions, including (but not limited to) such proposed effects as “coupling between conformational and chemical motions”, “landscape searches” and “entropy funnels”. It is shown that none of these proposed effects have been experimentally demonstrated to contribute to catalysis, nor are they supported by consistent theoretical studies. On the other hand, it is clarified that careful simulation studies have excluded most (if not all) dynamical proposals. This review places significant emphasis on clarifying the role of logical definitions of different catalytic proposals, and on the need for a clear formulation in terms of the assumed potential surface and reaction coordinate. Finally, it is pointed out that electrostatic preorganization actually accounts for the observed catalytic effects of enzymes, through the corresponding changes in the activation free energies. PMID:20099310

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

PubMed

Horrell, Sam; Kekilli, Demet; Sen, Kakali; Owen, Robin L; Dworkowski, Florian S N; Antonyuk, Svetlana V; Keal, Thomas W; Yong, Chin W; Eady, Robert R; Hasnain, S Samar; Strange, Richard W; Hough, Michael A

2018-05-01

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

PubMed

Yokom, Adam L; Morishima, Yoshihiro; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R

2014-06-13

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy

PubMed Central

Vallurupalli, Pramodh; Hansen, D. Flemming; Kay, Lewis E.

2008-01-01

Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)–ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of 15N, 1HN, 13Cα, and 13CO chemical shifts, along with 1HN-15N, 1Hα-13Cα, and 1HN-13CO residual dipolar couplings and 13CO residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with 1HN-15N residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins. PMID:18701719

Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected ¹³C CPMG relaxation dispersion.

PubMed

Weininger, Ulrich; Respondek, Michal; Akke, Mikael

2012-09-01

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.

Maintaining and Enhancing Diversity of Sampled Protein Conformations in Robotics-Inspired Methods.

PubMed

Abella, Jayvee R; Moll, Mark; Kavraki, Lydia E

2018-01-01

The ability to efficiently sample structurally diverse protein conformations allows one to gain a high-level view of a protein's energy landscape. Algorithms from robot motion planning have been used for conformational sampling, and several of these algorithms promote diversity by keeping track of "coverage" in conformational space based on the local sampling density. However, large proteins present special challenges. In particular, larger systems require running many concurrent instances of these algorithms, but these algorithms can quickly become memory intensive because they typically keep previously sampled conformations in memory to maintain coverage estimates. In addition, robotics-inspired algorithms depend on defining useful perturbation strategies for exploring the conformational space, which is a difficult task for large proteins because such systems are typically more constrained and exhibit complex motions. In this article, we introduce two methodologies for maintaining and enhancing diversity in robotics-inspired conformational sampling. The first method addresses algorithms based on coverage estimates and leverages the use of a low-dimensional projection to define a global coverage grid that maintains coverage across concurrent runs of sampling. The second method is an automatic definition of a perturbation strategy through readily available flexibility information derived from B-factors, secondary structure, and rigidity analysis. Our results show a significant increase in the diversity of the conformations sampled for proteins consisting of up to 500 residues when applied to a specific robotics-inspired algorithm for conformational sampling. The methodologies presented in this article may be vital components for the scalability of robotics-inspired approaches.

Low Cost Solar Array Project: Composition Measurements by Analytical Photo Catalysis

NASA Technical Reports Server (NTRS)

Sutton, D. G.; Galvan, L.; Melzer, J.; Heidner, R. F., III

1979-01-01

The applicability of the photon catalysis technique for effecting composition analysis of silicon samples is discussed. A detector for the impurities Al, Cr, Fe, Mn, Ti, V, Mo and Zr is evaluated. During the first reporting period Al, Cr, Fe, and Mn were detected with the photon catalysis method. The best fluorescence lines to monitor and determine initial sensitivities to each of these elements by atomic absorption calibration were established. In the course of these tests vapor pressure curves for these four pure substances were also mapped. Ti and Si were detected. The best lines to monitor were catalogued and vapor pressure curves were determined. Attempts to detect vanadium were unsuccessful due to the refractory nature of this element and the limited temperature range of the evaporator.

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

PubMed

Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

2016-06-28

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

PubMed Central

2011-01-01

Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR. PMID:22060818

The influence of shale depositional fabric on the kinetics of hydrocarbon generation through control of mineral surface contact area on clay catalysis

NASA Astrophysics Data System (ADS)

Rahman, Habibur M.; Kennedy, Martin; Löhr, Stefan; Dewhurst, David N.; Sherwood, Neil; Yang, Shengyu; Horsfield, Brian

2018-01-01

Accurately assessing the temperature and hence the depth and timing of hydrocarbon generation is a critical step in the characterization of a petroleum system. Clay catalysis is a potentially significant modifier of hydrocarbon generation temperature, but experimental studies of clay catalysis show inconsistent or contradictory results. This study tests the hypothesis that source rock fabric itself is an influence on clay mineral catalysis as it controls the extent to which organic matter and clay minerals are physically associated. Two endmember clay-organic fabrics distinguish the source rocks studied: (1) a particulate fabric where organic matter is present as discrete, >5 μm particles and (2) a nanocomposite fabric in which amorphous organic matter is associated with clay mineral surfaces at sub-micron scale. High-resolution electron imaging and bulk geochemical characterisation confirm that samples of the Miocene Monterey Formation (California) are representative of the nanocomposite source rock endmember, whereas samples from the Permian Stuart Range Formation (South Australia) represent the particulate source rock endmember. Kinetic experiments are performed on paired whole rock and kerogen isolate samples from these two formations using open system, non-isothermal pyrolysis at three different heating rates (0.7, 2 and 5 K/min) to determine the effects of the different shale fabrics on hydrocarbon generation kinetics. Extrapolation to a modelled geological heating rate shows a 20 °C reduction in the onset temperature of hydrocarbon generation in Monterey Formation whole rock samples relative to paired kerogen isolates. This result is consistent with the Monterey Formations's nanocomposite fabric where clay catalysis can proceed because reactive clay minerals are intimately associated with organic matter. By contrast, there is no significant difference in the modelled hydrocarbon generation temperature of paired whole rock and kerogen isolates from the Stuart Range Formation. This is consistent with its particulate fabric, where relatively large, discrete organic particles have limited contact with the mineral matrix and the clay minerals are mainly diagenetic and physically segregated within pores. While heating rate may have a control on mineral matrix effects, this result shows that the extent to which organic matter and clay minerals are physically associated could have a significant effect on the timing of hydrocarbon generation, and is a function of the depositional environment and detrital vs diagenetic origin of clay minerals in source rocks.

PubChem3D: Conformer generation

PubMed Central

2011-01-01

Background PubChem, an open archive for the biological activities of small molecules, provides search and analysis tools to assist users in locating desired information. Many of these tools focus on the notion of chemical structure similarity at some level. PubChem3D enables similarity of chemical structure 3-D conformers to augment the existing similarity of 2-D chemical structure graphs. It is also desirable to relate theoretical 3-D descriptions of chemical structures to experimental biological activity. As such, it is important to be assured that the theoretical conformer models can reproduce experimentally determined bioactive conformations. In the present study, we investigate the effects of three primary conformer generation parameters (the fragment sampling rate, the energy window size, and force field variant) upon the accuracy of theoretical conformer models, and determined optimal settings for PubChem3D conformer model generation and conformer sampling. Results Using the software package OMEGA from OpenEye Scientific Software, Inc., theoretical 3-D conformer models were generated for 25,972 small-molecule ligands, whose 3-D structures were experimentally determined. Different values for primary conformer generation parameters were systematically tested to find optimal settings. Employing a greater fragment sampling rate than the default did not improve the accuracy of the theoretical conformer model ensembles. An ever increasing energy window did increase the overall average accuracy, with rapid convergence observed at 10 kcal/mol and 15 kcal/mol for model building and torsion search, respectively; however, subsequent study showed that an energy threshold of 25 kcal/mol for torsion search resulted in slightly improved results for larger and more flexible structures. Exclusion of coulomb terms from the 94s variant of the Merck molecular force field (MMFF94s) in the torsion search stage gave more accurate conformer models at lower energy windows. Overall average accuracy of reproduction of bioactive conformations was remarkably linear with respect to both non-hydrogen atom count ("size") and effective rotor count ("flexibility"). Using these as independent variables, a regression equation was developed to predict the RMSD accuracy of a theoretical ensemble to reproduce bioactive conformations. The equation was modified to give a minimum RMSD conformer sampling value to help ensure that 90% of the sampled theoretical models should contain at least one conformer within the RMSD sampling value to a "bioactive" conformation. Conclusion Optimal parameters for conformer generation using OMEGA were explored and determined. An equation was developed that provides an RMSD sampling value to use that is based on the relative accuracy to reproduce bioactive conformations. The optimal conformer generation parameters and RMSD sampling values determined are used by the PubChem3D project to generate theoretical conformer models. PMID:21272340

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

PubMed

Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

2015-08-01

F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1. © 2015 FEBS.

Toward accurate prediction of pKa values for internal protein residues: the importance of conformational relaxation and desolvation energy.

PubMed

Wallace, Jason A; Wang, Yuhang; Shi, Chuanyin; Pastoor, Kevin J; Nguyen, Bao-Linh; Xia, Kai; Shen, Jana K

2011-12-01

Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pK(a) prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pK(a) predictions but also inform about the physical origins of pK(a) shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pK(a) prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pK(a) values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pK(a)'s with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pK(a) values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pK(a) predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy. Copyright © 2011 Wiley-Liss, Inc.

Relating protein conformational changes to packing efficiency and disorder

PubMed Central

Bhardwaj, Nitin; Gerstein, Mark

2009-01-01

Changes in protein conformation play key roles in facilitating various biochemical processes, ranging from signaling and phosphorylation to transport and catalysis. While various factors that drive these motions such as environmental changes and binding of small molecules are well understood, specific causative effects on the structural features of the protein due to these conformational changes have not been studied on a large scale. Here, we study protein conformational changes in relation to two key structural metrics: packing efficiency and disorder. Packing has been shown to be crucial for protein stability and function by many protein design and engineering studies. We study changes in packing efficiency during conformational changes, thus extending the analysis from a static context to a dynamic perspective and report some interesting observations. First, we study various proteins that adopt alternate conformations and find that tendencies to show motion and change in packing efficiency are correlated: residues that change their packing efficiency show larger motions. Second, our results suggest that residues that show higher changes in packing during motion are located on the changing interfaces which are formed during these conformational changes. These changing interfaces are slightly different from shear or static interfaces that have been analyzed in previous studies. Third, analysis of packing efficiency changes in the context of secondary structure shows that, as expected, residues buried in helices show the least change in packing efficiency, whereas those embedded in bends are most likely to change packing. Finally, by relating protein disorder to motions, we show that marginally disordered residues which are ordered enough to be crystallized but have sequence patterns indicative of disorder show higher dislocation and a higher change in packing than ordered ones and are located mostly on the changing interfaces. Overall, our results demonstrate that between the two conformations, the cores of the proteins remain mostly intact, whereas the interfaces display the most elasticity, both in terms of disorder and change in packing efficiency. By doing a variety of tests, we also show that our observations are robust to the solvation state of the proteins. PMID:19472340

Correlated motion and the effect of distal mutations in dihydrofolate reductase

PubMed Central

Rod, Thomas H.; Radkiewicz, Jennifer L.; Brooks, Charles L.

2003-01-01

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618–12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks. PMID:12756296

Nicholas Cleveland | NREL

Science.gov Websites

tools for complex sample analysis Affiliated Research Programs Biochemical Catalysis Working Group The synthesis Catalyst characterization Catalyst testing and reaction screening Analysis of complex organics

Enhanced Ligand Sampling for Relative Protein–Ligand Binding Free Energy Calculations

PubMed Central

2016-01-01

Free energy calculations are used to study how strongly potential drug molecules interact with their target receptors. The accuracy of these calculations depends on the accuracy of the molecular dynamics (MD) force field as well as proper sampling of the major conformations of each molecule. However, proper sampling of ligand conformations can be difficult when there are large barriers separating the major ligand conformations. An example of this is for ligands with an asymmetrically substituted phenyl ring, where the presence of protein loops hinders the proper sampling of the different ring conformations. These ring conformations become more difficult to sample when the size of the functional groups attached to the ring increases. The Adaptive Integration Method (AIM) has been developed, which adaptively changes the alchemical coupling parameter λ during the MD simulation so that conformations sampled at one λ can aid sampling at the other λ values. The Accelerated Adaptive Integration Method (AcclAIM) builds on AIM by lowering potential barriers for specific degrees of freedom at intermediate λ values. However, these methods may not work when there are very large barriers separating the major ligand conformations. In this work, we describe a modification to AIM that improves sampling of the different ring conformations, even when there is a very large barrier between them. This method combines AIM with conformational Monte Carlo sampling, giving improved convergence of ring populations and the resulting free energy. This method, called AIM/MC, is applied to study the relative binding free energy for a pair of ligands that bind to thrombin and a different pair of ligands that bind to aspartyl protease β-APP cleaving enzyme 1 (BACE1). These protein–ligand binding free energy calculations illustrate the improvements in conformational sampling and the convergence of the free energy compared to both AIM and AcclAIM. PMID:25906170

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

PubMed

Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

2012-06-08

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Mechanistic Strategies for Catalysis Adopted by Evolutionary Distinct Family 43 Arabinanases*

PubMed Central

Santos, Camila R.; Polo, Carla C.; Costa, Maria C. M. F.; Nascimento, Andrey F. Z.; Meza, Andreia N.; Cota, Junio; Hoffmam, Zaira B.; Honorato, Rodrigo V.; Oliveira, Paulo S. L.; Goldman, Gustavo H.; Gilbert, Harry J.; Prade, Rolf A.; Ruller, Roberto; Squina, Fabio M.; Wong, Dominic W. S.; Murakami, Mário T.

2014-01-01

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked l-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg203–Ala230 loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation. PMID:24469445

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

PubMed

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Adsorption and conformational modification of fibronectin and fibrinogen adsorbed on hydroxyapatite. A QCM-D study.

PubMed

Fernández-Montes Moraleda, Belén; San Román, Julio; Rodríguez-Lorenzo, Luís M

2016-10-01

Hydroxyapatite is a bioactive ceramic frequently used for bone engineering/replacement. One of the parameters that influence the biological response to implanted materials is the conformation of the first adsorbed protein layer. In this work, the adsorption and conformational changes of two fibroid serum proteins; fibronectin and fibrinogen adsorbed onto four different hydroxyapatite powders are studied with a Quartz Crystal Microbalance with Dissipation (QCM-D). Each of the calcined apatites adsorbs less protein than their corresponding synthesized samples. Adsorption on synthesized samples yields always an extended conformation whereas a reorganization of the layer is observed for the calcined samples. Fg acquires a "Side on" conformation in all the samples at the beginning of the experiment except for one of the synthesized samples where an "End-on" conformation is obtained during the whole experiment. The Extended conformation is the active conformation for Fn. This conformation is favored by apatites with large specific surface area (SSA) and on highly concentrated media. Apatite surface features should be considered in the selection or design of materials for bone regeneration, since it is possible to control the conformation mode of attachment of Fn and Fg by an appropriate selection of them. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2585-2594, 2016. © 2016 Wiley Periodicals, Inc.

Conformational responses to changes in the state of ionization of titrable groups in proteins

NASA Astrophysics Data System (ADS)

Richman, Daniel Eric

Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction, and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pK a values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous p Ka values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins. Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly. Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H-bond) breaking on at least the mus timescale, and segmental unfolding were detected near titrating groups as pH decreased into the acidic range. The study identified local structural features and stabilities that modulate the magnitude of electrostatic effects. The data demonstrate that computational approaches to pK a calculations for surface groups must account for local fluctuations spanning a wide range of timescales. A comparative NMR spectroscopy study with the L25K and L125K variants of SNase, each with a Lys residue buried in the hydrophobic interior of the protein, determined locations, timescales, and amplitudes of backbone conformational reorganization coupled with ionization of the buried Lys residues. The L25K protein exhibited an ensemble of local fluctuations of the beta barrel in the hundreds of mus timescale and an ensemble of subglobally unfolded beta-barrel states in the hundreds of ms timescale with strong pH dependence. The L125K protein exhibited fluctuations of the helix around site 125 in the mus timescale, with negligible pH dependence. These data illustrate the diverse timescales and local structural properties of conformational reorganization coupled to ionization of buried groups, and the challenge to structure-based electrostatics calculations, which must capture these long-timescale processes.

Mapping Conformational Dynamics of Proteins Using Torsional Dynamics Simulations

PubMed Central

Gangupomu, Vamshi K.; Wagner, Jeffrey R.; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-01-01

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein’s experimentally established conformational substates. Conformational transition of calmodulin from the Ca2+-bound to the Ca2+-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. PMID:23663843

Mapping conformational dynamics of proteins using torsional dynamics simulations.

PubMed

Gangupomu, Vamshi K; Wagner, Jeffrey R; Park, In-Hee; Jain, Abhinandan; Vaidehi, Nagarajan

2013-05-07

All-atom molecular dynamics simulations are widely used to study the flexibility of protein conformations. However, enhanced sampling techniques are required for simulating protein dynamics that occur on the millisecond timescale. In this work, we show that torsional molecular dynamics simulations enhance protein conformational sampling by performing conformational search in the low-frequency torsional degrees of freedom. In this article, we use our recently developed torsional-dynamics method called Generalized Newton-Euler Inverse Mass Operator (GNEIMO) to study the conformational dynamics of four proteins. We investigate the use of the GNEIMO method in simulations of the conformationally flexible proteins fasciculin and calmodulin, as well as the less flexible crambin and bovine pancreatic trypsin inhibitor. For the latter two proteins, the GNEIMO simulations with an implicit-solvent model reproduced the average protein structural fluctuations and sample conformations similar to those from Cartesian simulations with explicit solvent. The application of GNEIMO with replica exchange to the study of fasciculin conformational dynamics produced sampling of two of this protein's experimentally established conformational substates. Conformational transition of calmodulin from the Ca(2+)-bound to the Ca(2+)-free conformation occurred readily with GNEIMO simulations. Moreover, the GNEIMO method generated an ensemble of conformations that satisfy about half of both short- and long-range interresidue distances obtained from NMR structures of holo to apo transitions in calmodulin. Although unconstrained all-atom Cartesian simulations have failed to sample transitions between the substates of fasciculin and calmodulin, GNEIMO simulations show the transitions in both systems. The relatively short simulation times required to capture these long-timescale conformational dynamics indicate that GNEIMO is a promising molecular-dynamics technique for studying domain motion in proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

PubMed Central

Liu, Yi-Liang; Tsai, I-Chen; Chang, Chia-Wei; Liao, Ya-Fan; Liu, Guang-Yaw; Hung, Hui-Chih

2013-01-01

This study investigated the functional roles of the N-terminal Ca2+ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca2+-binding site of PAD4 were mutated to disrupt the binding of Ca2+ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k cat/K m,BAEE values were 0.02, 0.63 and 0.01 s−1mM−1 (20.8 s−1mM−1 for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k cat value of 0.3 s−1 (13.3 s−1 for wild-type), whereas D176A retained some catalytic power with a k cat of 9.7 s−1. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k cat/K m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca2+ indicated that the conformational stability of the enzyme is highly dependent on Ca2+ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca2+ ions in the N-terminal Ca2+-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca2+ ions play critical roles in the full activation of the PAD4 enzyme. PMID:23382808

Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters

NASA Astrophysics Data System (ADS)

Landreh, Michael; Marklund, Erik G.; Uzdavinys, Povilas; Degiacomi, Matteo T.; Coincon, Mathieu; Gault, Joseph; Gupta, Kallol; Liko, Idlir; Benesch, Justin L. P.; Drew, David; Robinson, Carol V.

2017-01-01

Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.

Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

PubMed

Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

2016-03-01

Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively. © 2016 The Protein Society.

Molecular and Cellular Biophysics

NASA Astrophysics Data System (ADS)

Jackson, Meyer B.

2006-01-01

Molecular and Cellular Biophysics provides advanced undergraduate and graduate students with a foundation in the basic concepts of biophysics. Students who have taken physical chemistry and calculus courses will find this book an accessible and valuable aid in learning how these concepts can be used in biological research. The text provides a rigorous treatment of the fundamental theories in biophysics and illustrates their application with examples. Conformational transitions of proteins are studied first using thermodynamics, and subsequently with kinetics. Allosteric theory is developed as the synthesis of conformational transitions and association reactions. Basic ideas of thermodynamics and kinetics are applied to topics such as protein folding, enzyme catalysis and ion channel permeation. These concepts are then used as the building blocks in a treatment of membrane excitability. Through these examples, students will gain an understanding of the general importance and broad applicability of biophysical principles to biological problems. Offers a unique synthesis of concepts across a wide range of biophysical topics Provides a rigorous theoretical treatment, alongside applications in biological systems Author has been teaching biophysics for nearly 25 years

Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline.

PubMed

Christian, Thomas; Evilia, Caryn; Hou, Ya-Ming

2006-06-20

The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the kcat of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the Km and little effect on the Kd of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.

Mechanistic Insights from the Crystal Structure of Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase Complexed with the Adenylate Intermediate.

PubMed

Chen, Yaozong; Jiang, Yiping; Guo, Zhihong

2016-12-06

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, catalyzes an essential step in vitamin K biosynthesis and is a valuable drug target. Like many other adenylating enzymes, it changes its structure to accommodate substrate binding, catalysis, and product release along the path of a domain alternation catalytic mechanism. We have determined the crystal structure of its complex with the adenylation product, o-succinylbenzoyl-adenosine monophosphate (OSB-AMP), and captured a new postadenylation state. This structure presents unique features such as a strained conformation for the bound adenylate intermediate to indicate that it represents the enzyme state after completion of the adenylation reaction but before release of the C domain in its transition to the thioesterification conformation. By comparison to the ATP-bound preadenylation conformation, structural changes are identified in both the reactants and the active site to allow inference about how these changes accommodate and facilitate the adenylation reaction and to directly support an in-line backside attack nucleophilic substitution mechanism for the first half-reaction. Mutational analysis suggests that the conserved His196 plays an important role in desolvation of the active site rather than stabilizing the transition state of the adenylation reaction. In addition, comparison of the new structure with a previously determined OSB-AMP-bound structure of the same enzyme allows us to propose a release mechanism of the C domain in its alteration to form the thioesterification conformation. These findings allow us to better understand the domain alternation catalytic mechanism of MenE as well as many other adenylating enzymes.

Evolutionary-inspired probabilistic search for enhancing sampling of local minima in the protein energy surface

PubMed Central

2012-01-01

Background Despite computational challenges, elucidating conformations that a protein system assumes under physiologic conditions for the purpose of biological activity is a central problem in computational structural biology. While these conformations are associated with low energies in the energy surface that underlies the protein conformational space, few existing conformational search algorithms focus on explicitly sampling low-energy local minima in the protein energy surface. Methods This work proposes a novel probabilistic search framework, PLOW, that explicitly samples low-energy local minima in the protein energy surface. The framework combines algorithmic ingredients from evolutionary computation and computational structural biology to effectively explore the subspace of local minima. A greedy local search maps a conformation sampled in conformational space to a nearby local minimum. A perturbation move jumps out of a local minimum to obtain a new starting conformation for the greedy local search. The process repeats in an iterative fashion, resulting in a trajectory-based exploration of the subspace of local minima. Results and conclusions The analysis of PLOW's performance shows that, by navigating only the subspace of local minima, PLOW is able to sample conformations near a protein's native structure, either more effectively or as well as state-of-the-art methods that focus on reproducing the native structure for a protein system. Analysis of the actual subspace of local minima shows that PLOW samples this subspace more effectively that a naive sampling approach. Additional theoretical analysis reveals that the perturbation function employed by PLOW is key to its ability to sample a diverse set of low-energy conformations. This analysis also suggests directions for further research and novel applications for the proposed framework. PMID:22759582

Enhanced conformational sampling of carbohydrates by Hamiltonian replica-exchange simulation.

PubMed

Mishra, Sushil Kumar; Kara, Mahmut; Zacharias, Martin; Koca, Jaroslav

2014-01-01

Knowledge of the structure and conformational flexibility of carbohydrates in an aqueous solvent is important to improving our understanding of how carbohydrates function in biological systems. In this study, we extend a variant of the Hamiltonian replica-exchange molecular dynamics (MD) simulation to improve the conformational sampling of saccharides in an explicit solvent. During the simulations, a biasing potential along the glycosidic-dihedral linkage between the saccharide monomer units in an oligomer is applied at various levels along the replica runs to enable effective transitions between various conformations. One reference replica runs under the control of the original force field. The method was tested on disaccharide structures and further validated on biologically relevant blood group B, Lewis X and Lewis A trisaccharides. The biasing potential-based replica-exchange molecular dynamics (BP-REMD) method provided a significantly improved sampling of relevant conformational states compared with standard continuous MD simulations, with modest computational costs. Thus, the proposed BP-REMD approach adds a new dimension to existing carbohydrate conformational sampling approaches by enhancing conformational sampling in the presence of solvent molecules explicitly at relatively low computational cost.

Mg2+-dependent conformational changes and product release during DNA-catalyzed RNA ligation monitored by Bimane fluorescence

PubMed Central

Turriani, Elisa; Höbartner, Claudia; Jovin, Thomas M.

2015-01-01

Among the deoxyribozymes catalyzing the ligation of two RNA substrates, 7S11 generates a branched RNA containing a 2′,5′-linkage. We have attached the small fluorogenic probe Bimane to the triphosphate terminated RNA substrate and utilized emission intensity and anisotropy to follow structural rearrangements leading to a catalytically active complex upon addition of Mg2+. Bimane coupled to synthetic oligonucleotides is quenched by nearby guanines via photoinduced electron transfer. The degree of quenching is sensitive to changes in the base pairing of the residues involved and in their distances to the probe. These phenomena permit the characterization of various sequential processes in the assembly and function of 7S11: binding of Mg2+ to the triphosphate moiety, release of quenching of the probe by the 5′-terminal G residues of R-RNA as they engage in secondary base-pair interactions, local rearrangement into a distinct active conformation, and continuous release of the Bimane-labeled pyrophosphate during the course of reaction at 37°C. It was possible to assign equilibrium and rate constants and structural interpretations to the sequence of conformational transitions and catalysis, information useful for optimizing the design of next generation deoxyribozymes. The fluorescent signatures, thermodynamic equilibria and catalytic function of numerous mutated (base/substituted) molecules were examined. PMID:25505142

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

PubMed

Jacewicz, Agata; Trzemecka, Anna; Guja, Kip E; Plochocka, Danuta; Yakubovskaya, Elena; Bebenek, Anna; Garcia-Diaz, Miguel

2013-01-01

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Salt bridges: geometrically specific, designable interactions.

PubMed

Donald, Jason E; Kulp, Daniel W; DeGrado, William F

2011-03-01

Salt bridges occur frequently in proteins, providing conformational specificity and contributing to molecular recognition and catalysis. We present a comprehensive analysis of these interactions in protein structures by surveying a large database of protein structures. Salt bridges between Asp or Glu and His, Arg, or Lys display extremely well-defined geometric preferences. Several previously observed preferences are confirmed, and others that were previously unrecognized are discovered. Salt bridges are explored for their preferences for different separations in sequence and in space, geometric preferences within proteins and at protein-protein interfaces, co-operativity in networked salt bridges, inclusion within metal-binding sites, preference for acidic electrons, apparent conformational side chain entropy reduction on formation, and degree of burial. Salt bridges occur far more frequently between residues at close than distant sequence separations, but, at close distances, there remain strong preferences for salt bridges at specific separations. Specific types of complex salt bridges, involving three or more members, are also discovered. As we observe a strong relationship between the propensity to form a salt bridge and the placement of salt-bridging residues in protein sequences, we discuss the role that salt bridges might play in kinetically influencing protein folding and thermodynamically stabilizing the native conformation. We also develop a quantitative method to select appropriate crystal structure resolution and B-factor cutoffs. Detailed knowledge of these geometric and sequence dependences should aid de novo design and prediction algorithms. Copyright © 2010 Wiley-Liss, Inc.

Atomistic insights into regulatory mechanisms of the HER2 tyrosine kinase domain: a molecular dynamics study.

PubMed

Telesco, Shannon E; Radhakrishnan, Ravi

2009-03-18

HER2 (ErbB2/Neu) is a receptor tyrosine kinase belonging to the epidermal growth factor receptor (EGFR)/ErbB family and is overexpressed in 20-30% of human breast cancers. Although several crystal structures of ErbB kinases have been solved, the precise mechanism of HER2 activation remains unknown, and it has been suggested that HER2 is unique in its requirement for phosphorylation of Y877, a key tyrosine residue located in the activation loop. To elucidate mechanistic details of kinase domain regulation, we performed molecular dynamics simulations of a homology-modeled HER2 kinase structure in active and inactive conformations. Principal component analysis of the atomistic fluctuations reveals a tight coupling between the activation loop and catalytic loop that may contribute to alignment of residues required for catalysis in the active kinase. The free energy perturbation method is also employed to predict a role for phosphorylated Y877 in stabilizing the kinase conformations. Finally, simulation results are presented for a HER2/EGFR heterodimer and reveal that the dimeric interface induces a rearrangement of the alphaC helix toward the active conformation. Elucidation of the molecular regulatory mechanisms in HER2 will help establish structure-function relationships in the wild-type kinase, as well as predict mutations with a propensity for constitutive activation in HER2-mediated cancers.

Structural asymmetry and intersubunit communication in muscle creatine kinase.

PubMed

Ohren, Jeffrey F; Kundracik, Melisa L; Borders, Charles L; Edmiston, Paul; Viola, Ronald E

2007-03-01

The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65 A resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation.

Enhanced photochemical catalysis of TiO2 inverse opals by modification with ZnO or Fe2O3 using ALD and the hydrothermal method

NASA Astrophysics Data System (ADS)

Liu, Jiatong; Sun, Cuifeng; Fu, Ming; Long, Jie; He, Dawei; Wang, Yongsheng

2018-02-01

The development of porous materials exhibiting photon regulation abilities for improved photoelectrochemical catalysis performance is always one of the important goals of solar energy harvesting. In this study, methods to improve the photocatalytic activity of TiO2 inverse opals were discussed. TiO2 inverse opals were prepared by atomic layer deposition (ALD) using colloidal crystal templates. In addition, TiO2 inverse opal heterostructures were fabricated using colloidal heterocrystals by repeated vertical deposition using different colloidal spheres. The hydrothermal method and ALD were used to prepare ZnO- or Fe2O3-modified TiO2 inverse opals on the internal surfaces of the TiO2 porous structures. Although the photonic reflection band was not significantly varied by oxide modification, the presence of Fe2O3 in the TiO2 inverse opals enhanced their visible absorption. The conformally modified oxides on the TiO2 inverse opals could also form energy barriers and avoid the recombination of electrons and holes. The fabrication of the TiO2 photonic crystal heterostructures and modification with ZnO or Fe2O3 can enhance the photocatalytic activity of TiO2 inverse opals.

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model ofmore » the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.« less

Low operational stability of enzymes in dry organic solvents: changes in the active site might affect catalysis.

PubMed

Bansal, Vibha; Delgado, Yamixa; Legault, Marc; Barletta, Gabriel

2012-02-14

The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme's initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

Towards efficient chemical synthesis via engineering enzyme catalysis in biomimetic nanoreactors.

PubMed

Liu, Jia; Yang, Qihua; Li, Can

2015-09-18

Biocatalysis with immobilized enzymes as catalysts holds enormous promise in developing more efficient and sustainable processes for the synthesis of fine chemicals, chiral pharmaceuticals and biomass feedstocks. Despite the appealing potentials, nowadays the industrial-scale application of biocatalysts is still quite modest in comparison with that of traditional chemical catalysts. A critical issue is that the catalytic performance of enzymes, the sophisticated and vulnerable catalytic machineries, strongly depends on their intracellular working environment; however the working circumstances provided by the support matrix are radically different from those in cells. This often leads to various adverse consequences on enzyme conformation and dynamic properties, consequently decreasing the overall performance of immobilized enzymes with regard to their activity, selectivity and stability. Engineering enzyme catalysis in support nanopores by mimicking the physiological milieu of enzymes in vivo and investigating how the interior microenvironment of nanopores imposes an influence on enzyme behaviors in vitro are of paramount significance to modify and improve the catalytic functions of immobilized enzymes. In this feature article, we have summarized the recent advances in mimicking the working environment and working patterns of intracellular enzymes in nanopores of mesoporous silica-based supports. Especially, we have demonstrated that incorporation of polymers into silica nanopores could be a valuable approach to create the biomimetic microenvironment for enzymes in the immobilized state.

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds*

PubMed Central

Hehemann, Jan-Hendrik; Smyth, Leo; Yadav, Anuj; Vocadlo, David J.; Boraston, Alisdair B.

2012-01-01

Agars are abundant polysaccharides from marine red algae, and their chemical structure consists of alternating d-galactose and 3,6-anhydro-l-galactose residues, the latter of which are presumed to make the polymer recalcitrant to degradation by most terrestrial bacteria. Here we study a family 117 glycoside hydrolase (BpGH117) encoded within a recently discovered locus from the human gut bacterium Bacteroides plebeius. Consistent with this locus being involved in agarocolloid degradation, we show that BpGH117 is an exo-acting 3,6-anhydro-α-(1,3)-l-galactosidase that removes the 3,6-anhydrogalactose from the non-reducing end of neoagaro-oligosaccharides. A Michaelis complex of BpGH117 with neoagarobiose reveals the distortion of the constrained 3,6-anhydro-l-galactose into a conformation that favors catalysis. Furthermore, this complex, supported by analysis of site-directed mutants, provides evidence for an organization of the active site and positioning of the catalytic residues that are consistent with an inverting mechanism of catalysis and suggests that a histidine residue acts as the general acid. This latter feature differs from the vast majority of glycoside hydrolases, which use a carboxylic acid, highlighting the alternative strategies that enzymes may utilize in catalyzing the cleavage of glycosidic bonds. PMID:22393053

Methods in Enzymology: “Flexible backbone sampling methods to model and design protein alternative conformations”

PubMed Central

Ollikainen, Noah; Smith, Colin A.; Fraser, James S.; Kortemme, Tanja

2013-01-01

Sampling alternative conformations is key to understanding how proteins work and engineering them for new functions. However, accurately characterizing and modeling protein conformational ensembles remains experimentally and computationally challenging. These challenges must be met before protein conformational heterogeneity can be exploited in protein engineering and design. Here, as a stepping stone, we describe methods to detect alternative conformations in proteins and strategies to model these near-native conformational changes based on backrub-type Monte Carlo moves in Rosetta. We illustrate how Rosetta simulations that apply backrub moves improve modeling of point mutant side chain conformations, native side chain conformational heterogeneity, functional conformational changes, tolerated sequence space, protein interaction specificity, and amino acid co-variation across protein-protein interfaces. We include relevant Rosetta command lines and RosettaScripts to encourage the application of these types of simulations to other systems. Our work highlights that critical scoring and sampling improvements will be necessary to approximate conformational landscapes. Challenges for the future development of these methods include modeling conformational changes that propagate away from designed mutation sites and modulating backbone flexibility to predictively design functionally important conformational heterogeneity. PMID:23422426

Prediction of protein loop conformations using multiscale modeling methods with physical energy scoring functions.

PubMed

Olson, Mark A; Feig, Michael; Brooks, Charles L

2008-04-15

This article examines ab initio methods for the prediction of protein loops by a computational strategy of multiscale conformational sampling and physical energy scoring functions. Our approach consists of initial sampling of loop conformations from lattice-based low-resolution models followed by refinement using all-atom simulations. To allow enhanced conformational sampling, the replica exchange method was implemented. Physical energy functions based on CHARMM19 and CHARMM22 parameterizations with generalized Born (GB) solvent models were applied in scoring loop conformations extracted from the lattice simulations and, in the case of all-atom simulations, the ensemble of conformations were generated and scored with these models. Predictions are reported for 25 loop segments, each eight residues long and taken from a diverse set of 22 protein structures. We find that the simulations generally sampled conformations with low global root-mean-square-deviation (RMSD) for loop backbone coordinates from the known structures, whereas clustering conformations in RMSD space and scoring detected less favorable loop structures. Specifically, the lattice simulations sampled basins that exhibited an average global RMSD of 2.21 +/- 1.42 A, whereas clustering and scoring the loop conformations determined an RMSD of 3.72 +/- 1.91 A. Using CHARMM19/GB to refine the lattice conformations improved the sampling RMSD to 1.57 +/- 0.98 A and detection to 2.58 +/- 1.48 A. We found that further improvement could be gained from extending the upper temperature in the all-atom refinement from 400 to 800 K, where the results typically yield a reduction of approximately 1 A or greater in the RMSD of the detected loop. Overall, CHARMM19 with a simple pairwise GB solvent model is more efficient at sampling low-RMSD loop basins than CHARMM22 with a higher-resolution modified analytical GB model; however, the latter simulation method provides a more accurate description of the all-atom energy surface, yet demands a much greater computational cost. (c) 2007 Wiley Periodicals, Inc.

Turning Cucurbit[8]uril into a Supramolecular Nanoreactor for Asymmetric Catalysis

PubMed Central

Zheng, Lifei; Sonzini, Silvia; Ambarwati, Masyitha; Rosta, Edina

2015-01-01

Abstract Chiral macromolecules have been widely used as synthetic pockets to mimic natural enzymes and promote asymmetric reactions. An achiral host, cucurbit[8]uril (CB[8]), was used for an asymmetric Lewis acid catalyzed Diels–Alder reaction. We achieved a remarkable increase in enantioselectivity and a large rate acceleration in the presence of the nanoreactor by using an amino acid as the chiral source. Mechanistic and computational studies revealed that both the amino acid–Cu2+ complex and the dienophile substrate are included inside the macrocyclic host cavity, suggesting that contiguity and conformational constraints are fundamental to the catalytic process and rate enhancement. These results pave the way towards new studies on asymmetric reactions catalyzed in confined achiral cavities. PMID:27478269

Turning Cucurbit[8]uril into a Supramolecular Nanoreactor for Asymmetric Catalysis

PubMed Central

Zheng, Lifei; Sonzini, Silvia; Ambarwati, Masyitha; Rosta, Edina; Scherman, Oren A; Herrmann, Andreas

2015-01-01

Chiral macromolecules have been widely used as synthetic pockets to mimic natural enzymes and promote asymmetric reactions. An achiral host, cucurbit[8]uril (CB[8]), was used for an asymmetric Lewis acid catalyzed Diels–Alder reaction. We achieved a remarkable increase in enantioselectivity and a large rate acceleration in the presence of the nanoreactor by using an amino acid as the chiral source. Mechanistic and computational studies revealed that both the amino acid–Cu2+ complex and the dienophile substrate are included inside the macrocyclic host cavity, suggesting that contiguity and conformational constraints are fundamental to the catalytic process and rate enhancement. These results pave the way towards new studies on asymmetric reactions catalyzed in confined achiral cavities. PMID:26383272

Prediction of Stereochemistry using Q2MM

PubMed Central

2016-01-01

Conspectus The standard method of screening ligands for selectivity in asymmetric, transition metal-catalyzed reactions requires experimental testing of hundreds of ligands from ligand libraries. This “trial and error” process is costly in terms of time as well as resources and, in general, is scientifically and intellectually unsatisfying as it reveals little about the underlying mechanism behind the selectivity. The accurate computational prediction of stereoselectivity in enantioselective catalysis requires adequate conformational sampling of the selectivity-determining transition state but has to be fast enough to compete with experimental screening techniques to be useful for the synthetic chemist. Although electronic structure calculations are accurate and general, they are too slow to allow for sampling or fast screening of ligand libraries. The combined requirements can be fulfilled by using appropriately fitted transition state force fields (TSFFs) that represent the transition state as a minimum and allow fast conformational sampling using Monte Carlo. Quantum-guided molecular mechanics (Q2MM) is an automated force field parametrization method that generates accurate, reaction-specific TSFFs by fitting the functional form of an arbitrary force field using only electronic structure calculations by minimization of an objective function. A key feature that distinguishes the Q2MM method from many other automated parametrization procedures is the use of the Hessian matrix in addition to geometric parameters and relative energies. This alleviates the known problems of overfitting of TSFFs. After validation of the TSFF by comparison to electronic structure results for a test set and available experimental data, the stereoselectivity of a reaction can be calculated by summation over the Boltzman-averaged relative energies of the conformations leading to the different stereoisomers. The Q2MM method has been applied successfully to perform virtual ligand screens on a range of transition metal-catalyzed reactions that are important from both an industrial and an academic perspective. In this Account, we provide an overview of the continued improvement of the prediction of stereochemistry using Q2MM-derived TSFFs using four examples from different stages of development: (i) Pd-catalyzed allylation, (ii) OsO4-catalyzed asymmetric dihydroxylation (AD) of alkenes, (iii) Rh-catalyzed hydrogenation of enamides, and (iv) Ru-catalyzed hydrogenation of ketones. In the current form, correlation coefficients of 0.8–0.9 between calculated and experimental ee values are typical for a wide range of substrate–ligand combinations, and suitable ligands can be predicted for a given substrate with ∼80% accuracy. Although the generation of a TSFF requires an initial effort and will therefore be most useful for widely used reactions that require frequent screening campaigns, the method allows for a rapid virtual screen of large ligand libraries to focus experimental efforts on the most promising substrate–ligand combinations. PMID:27064579

Improvements to robotics-inspired conformational sampling in rosetta.

PubMed

Stein, Amelie; Kortemme, Tanja

2013-01-01

To accurately predict protein conformations in atomic detail, a computational method must be capable of sampling models sufficiently close to the native structure. All-atom sampling is difficult because of the vast number of possible conformations and extremely rugged energy landscapes. Here, we test three sampling strategies to address these difficulties: conformational diversification, intensification of torsion and omega-angle sampling and parameter annealing. We evaluate these strategies in the context of the robotics-based kinematic closure (KIC) method for local conformational sampling in Rosetta on an established benchmark set of 45 12-residue protein segments without regular secondary structure. We quantify performance as the fraction of sub-Angstrom models generated. While improvements with individual strategies are only modest, the combination of intensification and annealing strategies into a new "next-generation KIC" method yields a four-fold increase over standard KIC in the median percentage of sub-Angstrom models across the dataset. Such improvements enable progress on more difficult problems, as demonstrated on longer segments, several of which could not be accurately remodeled with previous methods. Given its improved sampling capability, next-generation KIC should allow advances in other applications such as local conformational remodeling of multiple segments simultaneously, flexible backbone sequence design, and development of more accurate energy functions.

Improvements to Robotics-Inspired Conformational Sampling in Rosetta

PubMed Central

Stein, Amelie; Kortemme, Tanja

2013-01-01

To accurately predict protein conformations in atomic detail, a computational method must be capable of sampling models sufficiently close to the native structure. All-atom sampling is difficult because of the vast number of possible conformations and extremely rugged energy landscapes. Here, we test three sampling strategies to address these difficulties: conformational diversification, intensification of torsion and omega-angle sampling and parameter annealing. We evaluate these strategies in the context of the robotics-based kinematic closure (KIC) method for local conformational sampling in Rosetta on an established benchmark set of 45 12-residue protein segments without regular secondary structure. We quantify performance as the fraction of sub-Angstrom models generated. While improvements with individual strategies are only modest, the combination of intensification and annealing strategies into a new “next-generation KIC” method yields a four-fold increase over standard KIC in the median percentage of sub-Angstrom models across the dataset. Such improvements enable progress on more difficult problems, as demonstrated on longer segments, several of which could not be accurately remodeled with previous methods. Given its improved sampling capability, next-generation KIC should allow advances in other applications such as local conformational remodeling of multiple segments simultaneously, flexible backbone sequence design, and development of more accurate energy functions. PMID:23704889

CAT-ACT—A new highly versatile x-ray spectroscopy beamline for catalysis and radionuclide science at the KIT synchrotron light facility ANKA

NASA Astrophysics Data System (ADS)

Zimina, A.; Dardenne, K.; Denecke, M. A.; Doronkin, D. E.; Huttel, E.; Lichtenberg, H.; Mangold, S.; Pruessmann, T.; Rothe, J.; Spangenberg, Th.; Steininger, R.; Vitova, T.; Geckeis, H.; Grunwaldt, J.-D.

2017-11-01

CAT-ACT—the hard X-ray beamline for CATalysis and ACTinide/radionuclide research at the KIT synchrotron radiation facility ANKA—is dedicated to X-ray spectroscopy, including "flux hungry" photon-in/photon-out and correlative techniques and combines state-of-the-art optics with a unique infrastructure for radionuclide and catalysis research. Measurements can be performed at photon energies varying between 3.4 keV and 55 keV, thus encompassing the actinide M- and L-edge or potassium K-edge up to the K-edges of the lanthanide series such as cerium. Well-established X-ray absorption fine structure spectroscopy in transmission and fluorescence detection modes is available in combination with high energy-resolution X-ray emission spectroscopy or X-ray diffraction techniques. The modular beamline design with two alternately operated in-line experimental stations enables sufficient flexibility to adapt sample environments and detection systems to many scientific challenges. The ACT experimental station focuses on various aspects of nuclear waste disposal within the mission of the Helmholtz association to contribute to the solution of one of the greatest scientific and social challenges of our time—the safe disposal of heat producing, highly radioactive waste forms from nuclear energy production. It augments present capabilities at the INE-Beamline by increasing the flux and extending the energy range into the hard X-ray regime. The CAT experimental station focuses on catalytic materials, e.g., for energy-related and exhaust gas catalysis. Characterization of catalytically active materials under realistic reaction conditions and the development of in situ and operando cells for sample environments close to industrial reactors are essential aspects at CAT.

CAT-ACT-A new highly versatile x-ray spectroscopy beamline for catalysis and radionuclide science at the KIT synchrotron light facility ANKA.

PubMed

Zimina, A; Dardenne, K; Denecke, M A; Doronkin, D E; Huttel, E; Lichtenberg, H; Mangold, S; Pruessmann, T; Rothe, J; Spangenberg, Th; Steininger, R; Vitova, T; Geckeis, H; Grunwaldt, J-D

2017-11-01

CAT-ACT-the hard X-ray beamline for CATalysis and ACTinide/radionuclide research at the KIT synchrotron radiation facility ANKA-is dedicated to X-ray spectroscopy, including "flux hungry" photon-in/photon-out and correlative techniques and combines state-of-the-art optics with a unique infrastructure for radionuclide and catalysis research. Measurements can be performed at photon energies varying between 3.4 keV and 55 keV, thus encompassing the actinide M- and L-edge or potassium K-edge up to the K-edges of the lanthanide series such as cerium. Well-established X-ray absorption fine structure spectroscopy in transmission and fluorescence detection modes is available in combination with high energy-resolution X-ray emission spectroscopy or X-ray diffraction techniques. The modular beamline design with two alternately operated in-line experimental stations enables sufficient flexibility to adapt sample environments and detection systems to many scientific challenges. The ACT experimental station focuses on various aspects of nuclear waste disposal within the mission of the Helmholtz association to contribute to the solution of one of the greatest scientific and social challenges of our time-the safe disposal of heat producing, highly radioactive waste forms from nuclear energy production. It augments present capabilities at the INE-Beamline by increasing the flux and extending the energy range into the hard X-ray regime. The CAT experimental station focuses on catalytic materials, e.g., for energy-related and exhaust gas catalysis. Characterization of catalytically active materials under realistic reaction conditions and the development of in situ and operando cells for sample environments close to industrial reactors are essential aspects at CAT.

Parallel cascade selection molecular dynamics for efficient conformational sampling and free energy calculation of proteins

NASA Astrophysics Data System (ADS)

Kitao, Akio; Harada, Ryuhei; Nishihara, Yasutaka; Tran, Duy Phuoc

2016-12-01

Parallel Cascade Selection Molecular Dynamics (PaCS-MD) was proposed as an efficient conformational sampling method to investigate conformational transition pathway of proteins. In PaCS-MD, cycles of (i) selection of initial structures for multiple independent MD simulations and (ii) conformational sampling by independent MD simulations are repeated until the convergence of the sampling. The selection is conducted so that protein conformation gradually approaches a target. The selection of snapshots is a key to enhance conformational changes by increasing the probability of rare event occurrence. Since the procedure of PaCS-MD is simple, no modification of MD programs is required; the selections of initial structures and the restart of the next cycle in the MD simulations can be handled with relatively simple scripts with straightforward implementation. Trajectories generated by PaCS-MD were further analyzed by the Markov state model (MSM), which enables calculation of free energy landscape. The combination of PaCS-MD and MSM is reported in this work.

Molecular Dynamics Study of the Opening Mechanism for DNA Polymerase I

PubMed Central

Miller, Bill R.; Parish, Carol A.; Wu, Eugene Y.

2014-01-01

During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme∶DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics. PMID:25474643

The structural basis of secondary active transport mechanisms.

PubMed

Forrest, Lucy R; Krämer, Reinhard; Ziegler, Christine

2011-02-01

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling. Copyright © 2010 Elsevier B.V. All rights reserved.

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

PubMed Central

Sengupta, Raghuvir N.; Van Schie, Sabine N.S.; Giambaşu, George; Dai, Qing; Yesselman, Joseph D.; York, Darrin; Piccirilli, Joseph A.; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH2) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed MC, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. PMID:26567314

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.

PubMed

Sengupta, Raghuvir N; Van Schie, Sabine N S; Giambaşu, George; Dai, Qing; Yesselman, Joseph D; York, Darrin; Piccirilli, Joseph A; Herschlag, Daniel

2016-01-01

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function. © 2015 Sengupta et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Leap-dynamics: efficient sampling of conformational space of proteins and peptides in solution.

PubMed

Kleinjung, J; Bayley, P; Fraternali, F

2000-03-31

A molecular simulation scheme, called Leap-dynamics, that provides efficient sampling of protein conformational space in solution is presented. The scheme is a combined approach using a fast sampling method, imposing conformational 'leaps' to force the system over energy barriers, and molecular dynamics (MD) for refinement. The presence of solvent is approximated by a potential of mean force depending on the solvent accessible surface area. The method has been successfully applied to N-acetyl-L-alanine-N-methylamide (alanine dipeptide), sampling experimentally observed conformations inaccessible to MD alone under the chosen conditions. The method predicts correctly the increased partial flexibility of the mutant Y35G compared to native bovine pancreatic trypsin inhibitor. In particular, the improvement over MD consists of the detection of conformational flexibility that corresponds closely to slow motions identified by nuclear magnetic resonance techniques.

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β-turns in peptide immunogenicity.

PubMed

Shukla, Rashmi Tambe; Sasidhar, Yellamraju U

2015-07-01

Earlier immunological experiments with a synthetic 36-residue peptide (75-110) from Influenza hemagglutinin have been shown to elicit anti-peptide antibodies (Ab) which could cross-react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide ((98)YPYDVPDYASLRS(110)) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS-AA/L all-atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β-turn formed by "DYAS" sequence) with greater preference at 310 K while, it samples a native-like conformation with relatively greater propensity at 293 K. The sequence "DYAS" samples β-turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β-turn involving "DYAS" sequence and in addition it also samples a β-turn formed by the sequence "YPYD" at its N-terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag-Ab recognition are discussed. © 2015 Wiley Periodicals, Inc.

Pyrolysis-catalysis of waste plastic using a nickel-stainless-steel mesh catalyst for high-value carbon products.

PubMed

Zhang, Yeshui; Nahil, Mohamad A; Wu, Chunfei; Williams, Paul T

2017-11-01

A stainless-steel mesh loaded with nickel catalyst was produced and used for the pyrolysis-catalysis of waste high-density polyethylene with the aim of producing high-value carbon products, including carbon nanotubes (CNTs). The catalysis temperature and plastic-to-catalyst ratio were investigated to determine the influence on the formation of different types of carbon deposited on the nickel-stainless-steel mesh catalyst. Increasing temperature from 700 to 900°C resulted in an increase in the carbon deposited on the nickel-loaded stainless-steel mesh catalyst from 32.5 to 38.0 wt%. The increase in sample-to-catalyst ratio reduced the amount of carbon deposited on the mesh catalyst in terms of g carbon g -1 plastic. The carbons were found to be largely composed of filamentous carbons, with negligible disordered (amorphous) carbons. Transmission electron microscopy analysis of the filamentous carbons revealed them to be composed of a large proportion (estimated at ∼40%) multi-walled carbon nanotubes (MWCNTs). The optimum process conditions for CNT production, in terms of yield and graphitic nature, determined by Raman spectroscopy, was catalysis temperature of 800°C and plastic-to-catalyst ratio of 1:2, where a mass of 334 mg of filamentous/MWCNTs g -1 plastic was produced.

Structural basis for catalysis at the membrane-water interface.

PubMed

Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B; Mancia, Filippo

2017-11-01

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

The comparison of automated clustering algorithms for resampling representative conformer ensembles with RMSD matrix.

PubMed

Kim, Hyoungrae; Jang, Cheongyun; Yadav, Dharmendra K; Kim, Mi-Hyun

2017-03-23

The accuracy of any 3D-QSAR, Pharmacophore and 3D-similarity based chemometric target fishing models are highly dependent on a reasonable sample of active conformations. Since a number of diverse conformational sampling algorithm exist, which exhaustively generate enough conformers, however model building methods relies on explicit number of common conformers. In this work, we have attempted to make clustering algorithms, which could find reasonable number of representative conformer ensembles automatically with asymmetric dissimilarity matrix generated from openeye tool kit. RMSD was the important descriptor (variable) of each column of the N × N matrix considered as N variables describing the relationship (network) between the conformer (in a row) and the other N conformers. This approach used to evaluate the performance of the well-known clustering algorithms by comparison in terms of generating representative conformer ensembles and test them over different matrix transformation functions considering the stability. In the network, the representative conformer group could be resampled for four kinds of algorithms with implicit parameters. The directed dissimilarity matrix becomes the only input to the clustering algorithms. Dunn index, Davies-Bouldin index, Eta-squared values and omega-squared values were used to evaluate the clustering algorithms with respect to the compactness and the explanatory power. The evaluation includes the reduction (abstraction) rate of the data, correlation between the sizes of the population and the samples, the computational complexity and the memory usage as well. Every algorithm could find representative conformers automatically without any user intervention, and they reduced the data to 14-19% of the original values within 1.13 s per sample at the most. The clustering methods are simple and practical as they are fast and do not ask for any explicit parameters. RCDTC presented the maximum Dunn and omega-squared values of the four algorithms in addition to consistent reduction rate between the population size and the sample size. The performance of the clustering algorithms was consistent over different transformation functions. Moreover, the clustering method can also be applied to molecular dynamics sampling simulation results.

Coupling Protein Dynamics with Proton Transport in Human Carbonic Anhydrase II

PubMed Central

2016-01-01

The role of protein dynamics in enzyme catalysis is one of the most highly debated topics in enzymology. The main controversy centers around what may be defined as functionally significant conformational fluctuations and how, if at all, these fluctuations couple to enzyme catalyzed events. To shed light on this debate, the conformational dynamics along the transition path surmounting the highest free energy barrier have been herein investigated for the rate limiting proton transport event in human carbonic anhydrase (HCA) II. Special attention has been placed on whether the motion of an excess proton is correlated with fluctuations in the surrounding protein and solvent matrix, which may be rare on the picosecond and subpicosecond time scales of molecular motions. It is found that several active site residues, which do not directly participate in the proton transport event, have a significant impact on the dynamics of the excess proton. These secondary participants are shown to strongly influence the active site environment, resulting in the creation of water clusters that are conducive to fast, moderately slow, or slow proton transport events. The identification and characterization of these secondary participants illuminates the role of protein dynamics in the catalytic efficiency of HCA II. PMID:27063577

Viral membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formedmore » draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.« less

The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

PubMed Central

Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

2014-01-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

PubMed

Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

2014-10-01

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition

PubMed Central

Zoll, Sebastian; Schlag, Martin; Shkumatov, Alexander V.; Rautenberg, Maren; Svergun, Dmitri I.; Götz, Friedrich

2012-01-01

The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open β-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division. PMID:22609916

Removal of slow-pulsing artifacts in in-phase 15N relaxation dispersion experiments using broadband 1H decoupling.

PubMed

Chatterjee, Soumya Deep; Ubbink, Marcellus; van Ingen, Hugo

2018-06-02

Understanding of the molecular mechanisms of protein function requires detailed insight into the conformational landscape accessible to the protein. Conformational changes can be crucial for biological processes, such as ligand binding, protein folding, and catalysis. NMR spectroscopy is exquisitely sensitive to such dynamic changes in protein conformations. In particular, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are a powerful tool to investigate protein dynamics on a millisecond time scale. CPMG experiments that probe the chemical shift modulation of 15 N in-phase magnetization are particularly attractive, due to their high sensitivity. These experiments require high power 1 H decoupling during the CPMG period to keep the 15 N magnetization in-phase. Recently, an improved version of the in-phase 15 N-CPMG experiment was introduced, offering greater ease of use by employing a single 1 H decoupling power for all CPMG pulsing rates. In these experiments however, incomplete decoupling of off-resonance amide 1 H spins introduces an artefactual dispersion of relaxation rates, the so-called slow-pulsing artifact. Here, we analyze the slow-pulsing artifact in detail and demonstrate that it can be suppressed through the use of composite pulse decoupling (CPD). We report the performances of various CPD schemes and show that CPD decoupling based on the 90 x -240 y -90 x element results in high-quality dispersion curves free of artifacts, even for amides with high 1 H offset.

Probing Conformational Changes of Human DNA Polymerase λ Using Mass Spectrometry-Based Protein Footprinting

PubMed Central

Fowler, Jason D.; Brown, Jessica A.; Kvaratskhelia, Mamuka; Suo, Zucai

2009-01-01

SUMMARY Crystallographic studies of the C-terminal, DNA polymerase β-like domain of human DNA polymerase lambda (fPolλ) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase β. To examine solution-phase protein conformation changes in fPolλ, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a Proline-rich domain at its N-terminus, we used a mass spectrometry - based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPolλ versus the binary complex of enzyme•gapped DNA and the ternary complex of enzyme•gapped DNA•dNTP. These experiments suggested that fPolλ does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide dNTP. Site-directed mutagenesis and pre-steady state kinetic studies confirmed the importance of R386 for the enzyme activity, and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases. PMID:19467241

Density-based clustering of small peptide conformations sampled from a molecular dynamics simulation.

PubMed

Kim, Minkyoung; Choi, Seung-Hoon; Kim, Junhyoung; Choi, Kihang; Shin, Jae-Min; Kang, Sang-Kee; Choi, Yun-Jaie; Jung, Dong Hyun

2009-11-01

This study describes the application of a density-based algorithm to clustering small peptide conformations after a molecular dynamics simulation. We propose a clustering method for small peptide conformations that enables adjacent clusters to be separated more clearly on the basis of neighbor density. Neighbor density means the number of neighboring conformations, so if a conformation has too few neighboring conformations, then it is considered as noise or an outlier and is excluded from the list of cluster members. With this approach, we can easily identify clusters in which the members are densely crowded in the conformational space, and we can safely avoid misclustering individual clusters linked by noise or outliers. Consideration of neighbor density significantly improves the efficiency of clustering of small peptide conformations sampled from molecular dynamics simulations and can be used for predicting peptide structures.

Beyond relationships between homogeneous and heterogeneous catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dixon, David A.; Katz, Alexander; Arslan, Ilke

2014-08-13

Scientists who regard catalysis as a coherent field have been striving for decades to articulate the fundamental unifying principles. But because these principles seem to be broader than chemistry, chemical engineering, and materials science combined, catalytic scientists commonly interact within the sub-domains of homogeneous, heterogeneous, and bio-catalysis, and increasingly within even narrower domains such as organocatalysis, phase-transfer catalysis, acid-base catalysis, zeolite catalysis, etc. Attempts to unify catalysis have motivated researchers to find relationships between homogeneous and heterogeneous catalysis and to mimic enzymes. These themes have inspired vibrant international meetings and workshops, and we have benefited from the idea exchanges andmore » have some thoughts about a path forward.« less

Cyndi: a multi-objective evolution algorithm based method for bioactive molecular conformational generation.

PubMed

Liu, Xiaofeng; Bai, Fang; Ouyang, Sisheng; Wang, Xicheng; Li, Honglin; Jiang, Hualiang

2009-03-31

Conformation generation is a ubiquitous problem in molecule modelling. Many applications require sampling the broad molecular conformational space or perceiving the bioactive conformers to ensure success. Numerous in silico methods have been proposed in an attempt to resolve the problem, ranging from deterministic to non-deterministic and systemic to stochastic ones. In this work, we described an efficient conformation sampling method named Cyndi, which is based on multi-objective evolution algorithm. The conformational perturbation is subjected to evolutionary operation on the genome encoded with dihedral torsions. Various objectives are designated to render the generated Pareto optimal conformers to be energy-favoured as well as evenly scattered across the conformational space. An optional objective concerning the degree of molecular extension is added to achieve geometrically extended or compact conformations which have been observed to impact the molecular bioactivity (J Comput -Aided Mol Des 2002, 16: 105-112). Testing the performance of Cyndi against a test set consisting of 329 small molecules reveals an average minimum RMSD of 0.864 A to corresponding bioactive conformations, indicating Cyndi is highly competitive against other conformation generation methods. Meanwhile, the high-speed performance (0.49 +/- 0.18 seconds per molecule) renders Cyndi to be a practical toolkit for conformational database preparation and facilitates subsequent pharmacophore mapping or rigid docking. The copy of precompiled executable of Cyndi and the test set molecules in mol2 format are accessible in Additional file 1. On the basis of MOEA algorithm, we present a new, highly efficient conformation generation method, Cyndi, and report the results of validation and performance studies comparing with other four methods. The results reveal that Cyndi is capable of generating geometrically diverse conformers and outperforms other four multiple conformer generators in the case of reproducing the bioactive conformations against 329 structures. The speed advantage indicates Cyndi is a powerful alternative method for extensive conformational sampling and large-scale conformer database preparation.

Peptidic Macrocycles - Conformational Sampling and Thermodynamic Characterization

PubMed Central

2018-01-01

Macrocycles are of considerable interest as highly specific drug candidates, yet they challenge standard conformer generators with their large number of rotatable bonds and conformational restrictions. Here, we present a molecular dynamics-based routine that bypasses current limitations in conformational sampling and extensively profiles the free energy landscape of peptidic macrocycles in solution. We perform accelerated molecular dynamics simulations to capture a diverse conformational ensemble. By applying an energetic cutoff, followed by geometric clustering, we demonstrate the striking robustness and efficiency of the approach in identifying highly populated conformational states of cyclic peptides. The resulting structural and thermodynamic information is benchmarked against interproton distances from NMR experiments and conformational states identified by X-ray crystallography. Using three different model systems of varying size and flexibility, we show that the method reliably reproduces experimentally determined structural ensembles and is capable of identifying key conformational states that include the bioactive conformation. Thus, the described approach is a robust method to generate conformations of peptidic macrocycles and holds promise for structure-based drug design. PMID:29652495

Peptidic Macrocycles - Conformational Sampling and Thermodynamic Characterization.

PubMed

Kamenik, Anna S; Lessel, Uta; Fuchs, Julian E; Fox, Thomas; Liedl, Klaus R

2018-05-29

Macrocycles are of considerable interest as highly specific drug candidates, yet they challenge standard conformer generators with their large number of rotatable bonds and conformational restrictions. Here, we present a molecular dynamics-based routine that bypasses current limitations in conformational sampling and extensively profiles the free energy landscape of peptidic macrocycles in solution. We perform accelerated molecular dynamics simulations to capture a diverse conformational ensemble. By applying an energetic cutoff, followed by geometric clustering, we demonstrate the striking robustness and efficiency of the approach in identifying highly populated conformational states of cyclic peptides. The resulting structural and thermodynamic information is benchmarked against interproton distances from NMR experiments and conformational states identified by X-ray crystallography. Using three different model systems of varying size and flexibility, we show that the method reliably reproduces experimentally determined structural ensembles and is capable of identifying key conformational states that include the bioactive conformation. Thus, the described approach is a robust method to generate conformations of peptidic macrocycles and holds promise for structure-based drug design.

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

PubMed Central

van Aalten, D. M. F.; Komander, D.; Synstad, B.; Gåseidnes, S.; Peter, M. G.; Eijsink, V. G. H.

2001-01-01

Chitinase B (ChiB) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites −2 to +3) shows closure of the “roof” of the active site tunnel. It also shows that the sugar in the −1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle. PMID:11481469

Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

PubMed Central

Vahidi, Siavash; Bi, Yumin; Dunn, Stanley D.; Konermann, Lars

2016-01-01

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling. PMID:26884184

Conformational sampling with stochastic proximity embedding and self-organizing superimposition: establishing reasonable parameters for their practical use.

PubMed

Tresadern, Gary; Agrafiotis, Dimitris K

2009-12-01

Stochastic proximity embedding (SPE) and self-organizing superimposition (SOS) are two recently introduced methods for conformational sampling that have shown great promise in several application domains. Our previous validation studies aimed at exploring the limits of these methods and have involved rather exhaustive conformational searches producing a large number of conformations. However, from a practical point of view, such searches have become the exception rather than the norm. The increasing popularity of virtual screening has created a need for 3D conformational search methods that produce meaningful answers in a relatively short period of time and work effectively on a large scale. In this work, we examine the performance of these algorithms and the effects of different parameter settings at varying levels of sampling. Our goal is to identify search protocols that can produce a diverse set of chemically sensible conformations and have a reasonable probability of sampling biologically active space within a small number of trials. Our results suggest that both SPE and SOS are extremely competitive in this regard and produce very satisfactory results with as few as 500 conformations per molecule. The results improve even further when the raw conformations are minimized with a molecular mechanics force field to remove minor imperfections and any residual strain. These findings provide additional evidence that these methods are suitable for many everyday modeling tasks, both high- and low-throughput.

γ-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications.

PubMed

Castellano, Immacolata; Merlino, Antonello

2012-10-01

γ-Glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are involved in glutathione metabolism and play critical roles in antioxidant defense, detoxification, and inflammation processes. Moreover, γ-GTs have been recently found to be involved in many physiological disorders, such as Parkinson's disease and diabetes. In this review, the main biochemical and structural properties of γ-GTs isolated from different sources, as well as their conformational stability and mechanism of catalysis, are described and examined with the aim of contributing to the discussion on their structure-function relationships. Possible applications of γ-glutamyltranspeptidases in different fields of biotechnology and medicine are also discussed.

Allosteric Inhibition of the Neuropeptidase Neurolysin*

PubMed Central

Hines, Christina S.; Ray, Kallol; Schmidt, Jack J.; Xiong, Fei; Feenstra, Rolf W.; Pras-Raves, Mia; de Moes, Jan Peter; Lange, Jos H. M.; Melikishvili, Manana; Fried, Michael G.; Mortenson, Paul; Charlton, Michael; Patel, Yogendra; Courtney, Stephen M.; Kruse, Chris G.; Rodgers, David W.

2014-01-01

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases. PMID:25378390

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non‐Heme Iron Oxidases

PubMed Central

McNeill, Luke A.; Brown, Toby J. N.; Sami, Malkit; Clifton, Ian J.; Burzlaff, Nicolai I.; Claridge, Timothy D. W.; Adlington, Robert M.; Baldwin, Jack E.

2017-01-01

Abstract Isopenicillin N synthase (IPNS) catalyses the four‐electron oxidation of a tripeptide, l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV), to give isopenicillin N (IPN), the first‐formed β‐lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co‐substrate. In the absence of substrate, the carbonyl oxygen of the side‐chain amide of the penultimate residue, Gln330, co‐ordinates to the active‐site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C‐terminal residues, which move to take up a conformation that extends the final α‐helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C‐terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side‐chains around the ACV binding pocket is important in catalysis. Deletion of seven C‐terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC‐MS and NMR analyses to be the ene‐thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β‐carbon of the other. A mechanism for its formation is proposed, supported by an X‐ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β‐carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non‐heme iron oxidases in general. PMID:28703303

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

PubMed

McNeill, Luke A; Brown, Toby J N; Sami, Malkit; Clifton, Ian J; Burzlaff, Nicolai I; Claridge, Timothy D W; Adlington, Robert M; Baldwin, Jack E; Rutledge, Peter J; Schofield, Christopher J

2017-09-18

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed β-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Efficient evaluation of sampling quality of molecular dynamics simulations by clustering of dihedral torsion angles and Sammon mapping.

PubMed

Frickenhaus, Stephan; Kannan, Srinivasaraghavan; Zacharias, Martin

2009-02-01

A direct conformational clustering and mapping approach for peptide conformations based on backbone dihedral angles has been developed and applied to compare conformational sampling of Met-enkephalin using two molecular dynamics (MD) methods. Efficient clustering in dihedrals has been achieved by evaluating all combinations resulting from independent clustering of each dihedral angle distribution, thus resolving all conformational substates. In contrast, Cartesian clustering was unable to accurately distinguish between all substates. Projection of clusters on dihedral principal component (PCA) subspaces did not result in efficient separation of highly populated clusters. However, representation in a nonlinear metric by Sammon mapping was able to separate well the 48 highest populated clusters in just two dimensions. In addition, this approach also allowed us to visualize the transition frequencies between clusters efficiently. Significantly, higher transition frequencies between more distinct conformational substates were found for a recently developed biasing-potential replica exchange MD simulation method allowing faster sampling of possible substates compared to conventional MD simulations. Although the number of theoretically possible clusters grows exponentially with peptide length, in practice, the number of clusters is only limited by the sampling size (typically much smaller), and therefore the method is well suited also for large systems. The approach could be useful to rapidly and accurately evaluate conformational sampling during MD simulations, to compare different sampling strategies and eventually to detect kinetic bottlenecks in folding pathways.

Network visualization of conformational sampling during molecular dynamics simulation.

PubMed

Ahlstrom, Logan S; Baker, Joseph Lee; Ehrlich, Kent; Campbell, Zachary T; Patel, Sunita; Vorontsov, Ivan I; Tama, Florence; Miyashita, Osamu

2013-11-01

Effective data reduction methods are necessary for uncovering the inherent conformational relationships present in large molecular dynamics (MD) trajectories. Clustering algorithms provide a means to interpret the conformational sampling of molecules during simulation by grouping trajectory snapshots into a few subgroups, or clusters, but the relationships between the individual clusters may not be readily understood. Here we show that network analysis can be used to visualize the dominant conformational states explored during simulation as well as the connectivity between them, providing a more coherent description of conformational space than traditional clustering techniques alone. We compare the results of network visualization against 11 clustering algorithms and principal component conformer plots. Several MD simulations of proteins undergoing different conformational changes demonstrate the effectiveness of networks in reaching functional conclusions. Copyright © 2013 Elsevier Inc. All rights reserved.

Exploring protein kinase conformation using swarm-enhanced sampling molecular dynamics.

PubMed

Atzori, Alessio; Bruce, Neil J; Burusco, Kepa K; Wroblowski, Berthold; Bonnet, Pascal; Bryce, Richard A

2014-10-27

Protein plasticity, while often linked to biological function, also provides opportunities for rational design of selective and potent inhibitors of their function. The application of computational methods to the prediction of concealed protein concavities is challenging, as the motions involved can be significant and occur over long time scales. Here we introduce the swarm-enhanced sampling molecular dynamics (sesMD) method as a tool to improve sampling of conformational landscapes. In this approach, a swarm of replica simulations interact cooperatively via a set of pairwise potentials incorporating attractive and repulsive components. We apply the sesMD approach to explore the conformations of the DFG motif in the protein p38α mitogen-activated protein kinase. In contrast to multiple MD simulations, sesMD trajectories sample a range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between the DFG-in and DFG-out conformations are predicted to have druggable pockets of interest for structure-based ligand design.

Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms☆

PubMed Central

Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

2016-01-01

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins. Guest Editors: J.C. Gumbart and Sergei Noskov. PMID:26766517

Crystal Structure of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 Å Resolution: Dynamics of Catalytic Residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oakley, Aaron J.; Klvana, Martin; Otyepka, Michal

We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 {angstrom} resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle (126{sup o}) that deviated by 13.4{sup o} from the 'ideal' bond anglemore » of 112.6{sup o}. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N{sub {delta}1} and Asp108 in conformation A gave the most exothermic reaction ({Delta}H = -22 kcal/mol). With His272 doubly protonated at N{sub {delta}1} and N{sub {epsilon}2}, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.« less

Pickett angles and Cremer-Pople coordinates as collective variables for the enhanced sampling of six-membered ring conformations

NASA Astrophysics Data System (ADS)

Sega, M.; Autieri, E.; Pederiva, F.

2011-01-01

Although completely equivalent for the description of puckered ring conformers, the two popular coordinates sets of Strauss-Pickett dihedral angles and Cremer-Pople spherical coordinates are shown to have contrasting features when employed as collective variables in free-energy calculations with accelerated sampling techniques. Results from a 100 ns molecular dynamics simulation at conformational equilibrium and from combined metadynamics/umbrella sampling calculations of glucose are exploited to elucidate these differences.

Enhanced conformational sampling using enveloping distribution sampling.

PubMed

Lin, Zhixiong; van Gunsteren, Wilfred F

2013-10-14

To lessen the problem of insufficient conformational sampling in biomolecular simulations is still a major challenge in computational biochemistry. In this article, an application of the method of enveloping distribution sampling (EDS) is proposed that addresses this challenge and its sampling efficiency is demonstrated in simulations of a hexa-β-peptide whose conformational equilibrium encompasses two different helical folds, i.e., a right-handed 2.7(10∕12)-helix and a left-handed 3(14)-helix, separated by a high energy barrier. Standard MD simulations of this peptide using the GROMOS 53A6 force field did not reach convergence of the free enthalpy difference between the two helices even after 500 ns of simulation time. The use of soft-core non-bonded interactions in the centre of the peptide did enhance the number of transitions between the helices, but at the same time led to neglect of relevant helical configurations. In the simulations of a two-state EDS reference Hamiltonian that envelops both the physical peptide and the soft-core peptide, sampling of the conformational space of the physical peptide ensures that physically relevant conformations can be visited, and sampling of the conformational space of the soft-core peptide helps to enhance the transitions between the two helices. The EDS simulations sampled many more transitions between the two helices and showed much faster convergence of the relative free enthalpy of the two helices compared with the standard MD simulations with only a slightly larger computational effort to determine optimized EDS parameters. Combined with various methods to smoothen the potential energy surface, the proposed EDS application will be a powerful technique to enhance the sampling efficiency in biomolecular simulations.

Enhanced conformational sampling using replica exchange with concurrent solute scaling and hamiltonian biasing realized in one dimension.

PubMed

Yang, Mingjun; Huang, Jing; MacKerell, Alexander D

2015-06-09

Replica exchange (REX) is a powerful computational tool for overcoming the quasi-ergodic sampling problem of complex molecular systems. Recently, several multidimensional extensions of this method have been developed to realize exchanges in both temperature and biasing potential space or the use of multiple biasing potentials to improve sampling efficiency. However, increased computational cost due to the multidimensionality of exchanges becomes challenging for use on complex systems under explicit solvent conditions. In this study, we develop a one-dimensional (1D) REX algorithm to concurrently combine the advantages of overall enhanced sampling from Hamiltonian solute scaling and the specific enhancement of collective variables using Hamiltonian biasing potentials. In the present Hamiltonian replica exchange method, termed HREST-BP, Hamiltonian solute scaling is applied to the solute subsystem, and its interactions with the environment to enhance overall conformational transitions and biasing potentials are added along selected collective variables associated with specific conformational transitions, thereby balancing the sampling of different hierarchical degrees of freedom. The two enhanced sampling approaches are implemented concurrently allowing for the use of a small number of replicas (e.g., 6 to 8) in 1D, thus greatly reducing the computational cost in complex system simulations. The present method is applied to conformational sampling of two nitrogen-linked glycans (N-glycans) found on the HIV gp120 envelope protein. Considering the general importance of the conformational sampling problem, HREST-BP represents an efficient procedure for the study of complex saccharides, and, more generally, the method is anticipated to be of general utility for the conformational sampling in a wide range of macromolecular systems.

Characterizing the Conformational Landscape of Flavivirus Fusion Peptides via Simulation and Experiment

PubMed Central

Marzinek, Jan K.; Lakshminarayanan, Rajamani; Goh, Eunice; Huber, Roland G.; Panzade, Sadhana; Verma, Chandra; Bond, Peter J.

2016-01-01

Conformational changes in the envelope proteins of flaviviruses help to expose the highly conserved fusion peptide (FP), a region which is critical to membrane fusion and host cell infection, and which represents a significant target for antiviral drugs and antibodies. In principle, extended timescale atomic-resolution simulations may be used to characterize the dynamics of such peptides. However, the resultant accuracy is critically dependent upon both the underlying force field and sufficient conformational sampling. In the present study, we report a comprehensive comparison of three simulation methods and four force fields comprising a total of more than 40 μs of sampling. Additionally, we describe the conformational landscape of the FP fold across all flavivirus family members. All investigated methods sampled conformations close to available X-ray structures, but exhibited differently populated ensembles. The best force field / sampling combination was sufficiently accurate to predict that the solvated peptide fold is less ordered than in the crystallographic state, which was subsequently confirmed via circular dichroism and spectrofluorometric measurements. Finally, the conformational landscape of a mutant incapable of membrane fusion was significantly shallower than wild-type variants, suggesting that dynamics should be considered when therapeutically targeting FP epitopes. PMID:26785994

Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain.

PubMed

Selwa, Edithe; Huynh, Tru; Ciccotti, Giovanni; Maragliano, Luca; Malliavin, Thérèse E

2014-10-01

The catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X-ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature-accelerated molecular dynamics (TAMD) simulations to generate putative all-atom models of globular conformations sampled by CaM-free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter-domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations. © 2014 Wiley Periodicals, Inc.

Conformational Sampling of a Biomolecular Rugged Energy Landscape.

PubMed

Rydzewski, Jakub; Jakubowski, Rafal; Nicosia, Giuseppe; Nowak, Wieslaw

2018-01-01

The protein structure refinement using conformational sampling is important in hitherto protein studies. In this paper, we examined the protein structure refinement by means of potential energy minimization using immune computing as a method of sampling conformations. The method was tested on the x-ray structure and 30 decoys of the mutant of [Leu]Enkephalin, a paradigmatic example of the biomolecular multiple-minima problem. In order to score the refined conformations, we used a standard potential energy function with the OPLSAA force field. The effectiveness of the search was assessed using a variety of methods. The robustness of sampling was checked by the energy yield function which measures quantitatively the number of the peptide decoys residing in an energetic funnel. Furthermore, the potential energy-dependent Pareto fronts were calculated to elucidate dissimilarities between peptide conformations and the native state as observed by x-ray crystallography. Our results showed that the probed potential energy landscape of [Leu]Enkephalin is self-similar on different metric scales and that the local potential energy minima of the peptide decoys are metastable, thus they can be refined to conformations whose potential energy is decreased by approximately 250 kJ/mol.

Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

PubMed

Lenz, Stefan A P; Wetmore, Stacey D

2016-02-09

Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

A normal mode-based geometric simulation approach for exploring biologically relevant conformational transitions in proteins.

PubMed

Ahmed, Aqeel; Rippmann, Friedrich; Barnickel, Gerhard; Gohlke, Holger

2011-07-25

A three-step approach for multiscale modeling of protein conformational changes is presented that incorporates information about preferred directions of protein motions into a geometric simulation algorithm. The first two steps are based on a rigid cluster normal-mode analysis (RCNMA). Low-frequency normal modes are used in the third step (NMSim) to extend the recently introduced idea of constrained geometric simulations of diffusive motions in proteins by biasing backbone motions of the protein, whereas side-chain motions are biased toward favorable rotamer states. The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. When applied to a data set of proteins with experimentally observed conformational changes, conformational variabilities are reproduced very well for 4 out of 5 proteins that show domain motions, with correlation coefficients r > 0.70 and as high as r = 0.92 in the case of adenylate kinase. In 7 out of 8 cases, NMSim simulations starting from unbound structures are able to sample conformations that are similar (root-mean-square deviation = 1.0-3.1 Å) to ligand bound conformations. An NMSim generated pathway of conformational change of adenylate kinase correctly describes the sequence of domain closing. The NMSim approach is a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or as starting points for more sophisticated sampling techniques.

Equilibrium Molecular Thermodynamics from Kirkwood Sampling

PubMed Central

2015-01-01

We present two methods for barrierless equilibrium sampling of molecular systems based on the recently proposed Kirkwood method (J. Chem. Phys.2009, 130, 134102). Kirkwood sampling employs low-order correlations among internal coordinates of a molecule for random (or non-Markovian) sampling of the high dimensional conformational space. This is a geometrical sampling method independent of the potential energy surface. The first method is a variant of biased Monte Carlo, where Kirkwood sampling is used for generating trial Monte Carlo moves. Using this method, equilibrium distributions corresponding to different temperatures and potential energy functions can be generated from a given set of low-order correlations. Since Kirkwood samples are generated independently, this method is ideally suited for massively parallel distributed computing. The second approach is a variant of reservoir replica exchange, where Kirkwood sampling is used to construct a reservoir of conformations, which exchanges conformations with the replicas performing equilibrium sampling corresponding to different thermodynamic states. Coupling with the Kirkwood reservoir enhances sampling by facilitating global jumps in the conformational space. The efficiency of both methods depends on the overlap of the Kirkwood distribution with the target equilibrium distribution. We present proof-of-concept results for a model nine-atom linear molecule and alanine dipeptide. PMID:25915525

Identification of Rare Lewis Oligosaccharide Conformers in Aqueous Solution Using Enhanced Sampling Molecular Dynamics.

PubMed

Alibay, Irfan; Burusco, Kepa K; Bruce, Neil J; Bryce, Richard A

2018-03-08

Determining the conformations accessible to carbohydrate ligands in aqueous solution is important for understanding their biological action. In this work, we evaluate the conformational free-energy surfaces of Lewis oligosaccharides in explicit aqueous solvent using a multidimensional variant of the swarm-enhanced sampling molecular dynamics (msesMD) method; we compare with multi-microsecond unbiased MD simulations, umbrella sampling, and accelerated MD approaches. For the sialyl Lewis A tetrasaccharide, msesMD simulations in aqueous solution predict conformer landscapes in general agreement with the other biased methods and with triplicate unbiased 10 μs trajectories; these simulations find a predominance of closed conformer and a range of low-occupancy open forms. The msesMD simulations also suggest closed-to-open transitions in the tetrasaccharide are facilitated by changes in ring puckering of its GlcNAc residue away from the 4 C 1 form, in line with previous work. For sialyl Lewis X tetrasaccharide, msesMD simulations predict a minor population of an open form in solution corresponding to a rare lectin-bound pose observed crystallographically. Overall, from comparison with biased MD calculations, we find that triplicate 10 μs unbiased MD simulations may not be enough to fully sample glycan conformations in aqueous solution. However, the computational efficiency and intuitive approach of the msesMD method suggest potential for its application in glycomics as a tool for analysis of oligosaccharide conformation.

Probing RNA Native Conformational Ensembles with Structural Constraints.

PubMed

Fonseca, Rasmus; van den Bedem, Henry; Bernauer, Julie

2016-05-01

Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature.

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

PubMed

Stranava, Martin; Man, Petr; Skálová, Tereza; Kolenko, Petr; Blaha, Jan; Fojtikova, Veronika; Martínek, Václav; Dohnálek, Jan; Lengalova, Alzbeta; Rosůlek, Michal; Shimizu, Toru; Martínková, Markéta

2017-12-22

The heme-based oxygen sensor histidine kinase Af GcHK is part of a two-component signal transduction system in bacteria. O 2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His 183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH - and -CN - complexes of Af GcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN - and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length Af GcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of Af GcHK. We conclude that Af GcHK functions as an ensemble of molecules sampling at least two conformational states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Distance geometry protocol to generate conformations of natural products to structurally interpret ion mobility-mass spectrometry collision cross sections.

PubMed

Stow, Sarah M; Goodwin, Cody R; Kliman, Michal; Bachmann, Brian O; McLean, John A; Lybrand, Terry P

2014-12-04

Ion mobility-mass spectrometry (IM-MS) allows the separation of ionized molecules based on their charge-to-surface area (IM) and mass-to-charge ratio (MS), respectively. The IM drift time data that is obtained is used to calculate the ion-neutral collision cross section (CCS) of the ionized molecule with the neutral drift gas, which is directly related to the ion conformation and hence molecular size and shape. Studying the conformational landscape of these ionized molecules computationally provides interpretation to delineate the potential structures that these CCS values could represent, or conversely, structural motifs not consistent with the IM data. A challenge in the IM-MS community is the ability to rapidly compute conformations to interpret natural product data, a class of molecules exhibiting a broad range of biological activity. The diversity of biological activity is, in part, related to the unique structural characteristics often observed for natural products. Contemporary approaches to structurally interpret IM-MS data for peptides and proteins typically utilize molecular dynamics (MD) simulations to sample conformational space. However, MD calculations are computationally expensive, they require a force field that accurately describes the molecule of interest, and there is no simple metric that indicates when sufficient conformational sampling has been achieved. Distance geometry is a computationally inexpensive approach that creates conformations based on sampling different pairwise distances between the atoms within the molecule and therefore does not require a force field. Progressively larger distance bounds can be used in distance geometry calculations, providing in principle a strategy to assess when all plausible conformations have been sampled. Our results suggest that distance geometry is a computationally efficient and potentially superior strategy for conformational analysis of natural products to interpret gas-phase CCS data.

Distance Geometry Protocol to Generate Conformations of Natural Products to Structurally Interpret Ion Mobility-Mass Spectrometry Collision Cross Sections

PubMed Central

2015-01-01

Ion mobility-mass spectrometry (IM-MS) allows the separation of ionized molecules based on their charge-to-surface area (IM) and mass-to-charge ratio (MS), respectively. The IM drift time data that is obtained is used to calculate the ion-neutral collision cross section (CCS) of the ionized molecule with the neutral drift gas, which is directly related to the ion conformation and hence molecular size and shape. Studying the conformational landscape of these ionized molecules computationally provides interpretation to delineate the potential structures that these CCS values could represent, or conversely, structural motifs not consistent with the IM data. A challenge in the IM-MS community is the ability to rapidly compute conformations to interpret natural product data, a class of molecules exhibiting a broad range of biological activity. The diversity of biological activity is, in part, related to the unique structural characteristics often observed for natural products. Contemporary approaches to structurally interpret IM-MS data for peptides and proteins typically utilize molecular dynamics (MD) simulations to sample conformational space. However, MD calculations are computationally expensive, they require a force field that accurately describes the molecule of interest, and there is no simple metric that indicates when sufficient conformational sampling has been achieved. Distance geometry is a computationally inexpensive approach that creates conformations based on sampling different pairwise distances between the atoms within the molecule and therefore does not require a force field. Progressively larger distance bounds can be used in distance geometry calculations, providing in principle a strategy to assess when all plausible conformations have been sampled. Our results suggest that distance geometry is a computationally efficient and potentially superior strategy for conformational analysis of natural products to interpret gas-phase CCS data. PMID:25360896

ClustENM: ENM-Based Sampling of Essential Conformational Space at Full Atomic Resolution

PubMed Central

Kurkcuoglu, Zeynep; Bahar, Ivet; Doruker, Pemra

2016-01-01

Accurate sampling of conformational space and, in particular, the transitions between functional substates has been a challenge in molecular dynamic (MD) simulations of large biomolecular systems. We developed an Elastic Network Model (ENM)-based computational method, ClustENM, for sampling large conformational changes of biomolecules with various sizes and oligomerization states. ClustENM is an iterative method that combines ENM with energy minimization and clustering steps. It is an unbiased technique, which requires only an initial structure as input, and no information about the target conformation. To test the performance of ClustENM, we applied it to six biomolecular systems: adenylate kinase (AK), calmodulin, p38 MAP kinase, HIV-1 reverse transcriptase (RT), triosephosphate isomerase (TIM), and the 70S ribosomal complex. The generated ensembles of conformers determined at atomic resolution show good agreement with experimental data (979 structures resolved by X-ray and/or NMR) and encompass the subspaces covered in independent MD simulations for TIM, p38, and RT. ClustENM emerges as a computationally efficient tool for characterizing the conformational space of large systems at atomic detail, in addition to generating a representative ensemble of conformers that can be advantageously used in simulating substrate/ligand-binding events. PMID:27494296

Magnetic beads-based DNAzyme recognition and AuNPs-based enzymatic catalysis amplification for visual detection of trace uranyl ion in aqueous environment.

PubMed

Zhang, Hongyan; Lin, Ling; Zeng, Xiaoxue; Ruan, Yajuan; Wu, Yongning; Lin, Minggui; He, Ye; Fu, FengFu

2016-04-15

We herein developed a novel biosensor for the visual detection of trace uranyl ion (UO2(2+)) in aqueous environment with high sensitivity and specificity by using DNAzyme-functionalized magnetic beads (MBs) for UO2(2+) recognition and gold nano-particles (AuNPs)-based enzymatic catalysis oxidation of TMB (3,3',5,5'-tetramethylbenzidine sulfate) for signal generation. The utilization of MBs facilitates the magnetic separation and collection of sensing system from complex sample solution, which leads to more convenient experimental operation and more strong resistibility of the biosensor to the matrix of sample, and the utilization of AuNPs-based enzymatic catalysis amplification greatly improved the sensitivity of the biosensor. Compared with the previous DNAzyme-based UO2(2+) sensors, the proposed biosensor has outstanding advantages such as relative high sensitivity and specificity, operation convenience, low cost and more strong resistibility to the matrix of sample. It can be used to detect as low as 0.02 ppb (74 pM) of UO2(2+) in aqueous environment by only naked-eye observation and 1.89 ppt (7.0 pM) of UO2(2+) by UV-visible spectrophotometer with a recovery of 93-99% and a RSD ≤ 5.0% (n=6) within 3h. Especially, the visual detection limit of 0.02 ppb (74 pM) is much lower than the maximum allowable level of UO2(2+) (130 nM) in the drinking water defined by the U.S. Environmental Protection Agency (EPA), indicating that our method meets the requirement of rapid and on-site detection of UO2(2+) in the aqueous environment by only naked-eye observation. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of a reaction cell for in-situ/operando studies of surface of a catalyst under a reaction condition and during catalysis.

PubMed

Nguyen, Luan; Tao, Franklin Feng

2016-06-01

Tracking surface chemistry of a catalyst during catalysis is significant for fundamental understanding of catalytic performance of the catalyst since it allows for establishing an intrinsic correlation between surface chemistry of a catalyst at its working status and its corresponding catalytic performance. Ambient pressure X-ray photoelectron spectroscopy can be used for in-situ studies of surfaces of different materials or devices in a gas. To simulate the gaseous environment of a catalyst in a fixed-bed a flowing gaseous environment of reactants around the catalyst is necessary. Here, we report the development of a new flowing reaction cell for simulating in-situ study of a catalyst surface under a reaction condition in gas of one reactant or during catalysis in a mixture of reactants of a catalytic reaction. The homemade reaction cell is installed in a high vacuum (HV) or ultrahigh vacuum (UHV) environment of a chamber. The flowing gas in the reaction cell is separated from the HV or UHV environment through well sealings at three interfaces between the reaction cell and X-ray window, sample door and aperture of front cone of an energy analyzer. Catalyst in the cell is heated through infrared laser beam introduced through a fiber optics interfaced with the reaction cell through a homemade feedthrough. The highly localized heating on the sample holder and Au-passivated internal surface of the reaction cell effectively minimizes any unwanted reactions potentially catalyzed by the reaction cell. The incorporated laser heating allows a fast heating and a high thermal stability of the sample at a high temperature. With this cell, a catalyst at 800 °C in a flowing gas can be tracked readily.

Direct sp(3)C-H acroleination of N-aryl-tetrahydroisoquinolines by merging photoredox catalysis with nucleophilic catalysis.

PubMed

Feng, Zhu-Jia; Xuan, Jun; Xia, Xu-Dong; Ding, Wei; Guo, Wei; Chen, Jia-Rong; Zou, You-Quan; Lu, Liang-Qiu; Xiao, Wen-Jing

2014-04-07

Sequence catalysis merging photoredox catalysis (PC) and nucleophilic catalysis (NC) has been realized for the direct sp(3) C-H acroleination of N-aryl-tetrahydroisoquinoline (THIQ). The reaction was performed under very mild conditions and afforded products in 50-91% yields. A catalytic asymmetric variant was proved to be successful with moderate enantioselectivities (up to 83 : 17 er).

The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

PubMed Central

Johnson, Troy A.; Holyoak, Todd

2012-01-01

Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

Elucidation of Kinetic Mechanisms of Human Translesion DNA Polymerase κ Using Tryptophan Mutants

PubMed Central

Zhao, Linlin; Pence, Matthew G.; Eoff, Robert L.; Yuan, Shuai; Fercu, Catinca A.; Guengerich, F. Peter

2014-01-01

In order to investigate the conformational dynamics of human DNA polymerase κ (hpol κ), we generated two mutants, Y50W (N-clasp region) and Y408W (linker between the thumb and little finger domains), using a Trp-null mutant (W214Y/W392H) of the hpol κ catalytic core enzyme. These mutants retained catalytic activity and similar patterns of selectivity for bypassing the DNA adduct 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), as judged by the results of steady-state and pre-steady-state kinetic experiments. Stopped-flow kinetic assays with hpol κ Y50W and T408W revealed a decrease in Trp fluorescence with the template G:dCTP pair but not for any mispairs. This decrease in fluorescence was not rate-limiting and is considered to be related to a conformational change necessary for correct nucleotidyl transfer. When a free 3′-hydroxyl was present on the primer, the Trp fluorescence returned to the baseline level at a rate similar to the observed kcat, suggesting that this change occurs during or after nucleotidyl transfer. However, polymerization rates (kpol) of extended-product formation were fast, indicating that the slow fluorescence step follows phosphodiester bond formation and is rate-limiting. Pyrophosphate formation and release were fast and are likely to precede the slower relaxation step. The available kinetic data were used to fit a simplified minimal model. The extracted rate constants confirmed that the conformational change after phosphodiester formation was rate-limiting for hpol κ catalysis with the template G:dCTP pair. PMID:25065501

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme.

PubMed

McCord, Lauren A; Liang, Wenguang G; Dowdell, Evan; Kalas, Vasilios; Hoey, Robert J; Koide, Akiko; Koide, Shohei; Tang, Wei-Jen

2013-08-20

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

Reactor for in situ measurements of spatially resolved kinetic data in heterogeneous catalysis

NASA Astrophysics Data System (ADS)

Horn, R.; Korup, O.; Geske, M.; Zavyalova, U.; Oprea, I.; Schlögl, R.

2010-06-01

The present work describes a reactor that allows in situ measurements of spatially resolved kinetic data in heterogeneous catalysis. The reactor design allows measurements up to temperatures of 1300 °C and 45 bar pressure, i.e., conditions of industrial relevance. The reactor involves reactants flowing through a solid catalyst bed containing a sampling capillary with a side sampling orifice through which a small fraction of the reacting fluid (gas or liquid) is transferred into an analytical device (e.g., mass spectrometer, gas chromatograph, high pressure liquid chromatograph) for quantitative analysis. The sampling capillary can be moved with μm resolution in or against flow direction to measure species profiles through the catalyst bed. Rotation of the sampling capillary allows averaging over several scan lines. The position of the sampling orifice is such that the capillary channel through the catalyst bed remains always occupied by the capillary preventing flow disturbance and fluid bypassing. The second function of the sampling capillary is to provide a well which can accommodate temperature probes such as a thermocouple or a pyrometer fiber. If a thermocouple is inserted in the sampling capillary and aligned with the sampling orifice fluid temperature profiles can be measured. A pyrometer fiber can be used to measure the temperature profile of the solid catalyst bed. Spatial profile measurements are demonstrated for methane oxidation on Pt and methane oxidative coupling on Li/MgO, both catalysts supported on reticulated α -Al2O3 foam supports.

Dual reactor for in situ/operando fluorescent mode XAS studies of sample containing low-concentration 3d or 5d metal elements

NASA Astrophysics Data System (ADS)

Nguyen, Luan; Tang, Yu; Li, Yuting; Zhang, Xiaoyan; Wang, Ding; Tao, Franklin Feng

2018-05-01

Transition metal elements are the most important elements of heterogeneous catalysts used for chemical and energy transformations. Many of these catalysts are active at a temperature higher than 400 °C. For a catalyst containing a 3d or 5d metal element with a low concentration, typically their released fluorescence upon the K-edge or L-edge adsorption of X-rays is collected for the analysis of chemical and coordination environments of these elements. However, it is challenging to perform in situ/operando X-ray absorption spectroscopy (XAS) studies of elements of low-energy absorption edges at a low concentration in a catalyst during catalysis at a temperature higher than about 450 °C. Here a unique reaction system consisting two reactors, called a dual reactor system, was designed for performing in situ or operando XAS studies of these elements of low-energy absorption edges in a catalyst at a low concentration during catalysis at a temperature higher than 450 °C in a fluorescent mode. This dual-reactor system contains a quartz reactor for preforming high-temperature catalysis up to 950 °C and a Kapton reactor remaining at a temperature up to 450 °C for collecting data in the same gas of catalysis. With this dual reactor, chemical and coordination environments of low-concentration metal elements with low-energy absorption edges such as the K-edge of 3d metals including Ti, V, Cr, Mn, Fe, Co, Ni, and Cu and L edge of 5d metals including W, Re, Os, Ir, Pt, and Au can be examined through first performing catalysis at a temperature higher than 450 °C in the quartz reactor and then immediately flipping the catalyst in the same gas flow to the Kapton reactor remained up to 450 °C to collect data. The capability of this dual reactor was demonstrated by tracking the Mn K-edge of the MnOx/Na2WO4 catalyst during activation in the temperature range of 300-900 °C and catalysis at 850 °C.

Effects of forcefield and sampling method in all-atom simulations of inherently disordered proteins: Application to conformational preferences of human amylin

PubMed Central

Peng, Enxi; Todorova, Nevena

2017-01-01

Although several computational modelling studies have investigated the conformational behaviour of inherently disordered protein (IDP) amylin, discrepancies in identifying its preferred solution conformations still exist between various forcefields and sampling methods used. Human islet amyloid polypeptide has long been a subject of research, both experimentally and theoretically, as the aggregation of this protein is believed to be the lead cause of type-II diabetes. In this work, we present a systematic forcefield assessment using one of the most advanced non-biased sampling techniques, Replica Exchange with Solute Tempering (REST2), by comparing the secondary structure preferences of monomeric amylin in solution. This study also aims to determine the ability of common forcefields to sample a transition of the protein from a helical membrane bound conformation into the disordered solution state of amylin. Our results demonstrated that the CHARMM22* forcefield showed the best ability to sample multiple conformational states inherent for amylin. It is revealed that REST2 yielded results qualitatively consistent with experiments and in quantitative agreement with other sampling methods, however far more computationally efficiently and without any bias. Therefore, combining an unbiased sampling technique such as REST2 with a vigorous forcefield testing could be suggested as an important step in developing an efficient and robust strategy for simulating IDPs. PMID:29023509

Effects of forcefield and sampling method in all-atom simulations of inherently disordered proteins: Application to conformational preferences of human amylin.

PubMed

Peng, Enxi; Todorova, Nevena; Yarovsky, Irene

2017-01-01

Although several computational modelling studies have investigated the conformational behaviour of inherently disordered protein (IDP) amylin, discrepancies in identifying its preferred solution conformations still exist between various forcefields and sampling methods used. Human islet amyloid polypeptide has long been a subject of research, both experimentally and theoretically, as the aggregation of this protein is believed to be the lead cause of type-II diabetes. In this work, we present a systematic forcefield assessment using one of the most advanced non-biased sampling techniques, Replica Exchange with Solute Tempering (REST2), by comparing the secondary structure preferences of monomeric amylin in solution. This study also aims to determine the ability of common forcefields to sample a transition of the protein from a helical membrane bound conformation into the disordered solution state of amylin. Our results demonstrated that the CHARMM22* forcefield showed the best ability to sample multiple conformational states inherent for amylin. It is revealed that REST2 yielded results qualitatively consistent with experiments and in quantitative agreement with other sampling methods, however far more computationally efficiently and without any bias. Therefore, combining an unbiased sampling technique such as REST2 with a vigorous forcefield testing could be suggested as an important step in developing an efficient and robust strategy for simulating IDPs.

An introduction to NMR-based approaches for measuring protein dynamics

PubMed Central

Kleckner, Ian R; Foster, Mark P

2010-01-01

Proteins are inherently flexible at ambient temperature. At equilibrium, they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biological functions including catalysis, binding, regulation and cellular structure. Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique for measuring these essential features of proteins. Here we provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common experimental and analytical framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape analysis, (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement. PMID:21059410

Geometrical criteria for characterizing open and closed states of WPD-loop in PTP1B

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Elizabeth Sobhia, M.

2012-06-01

Distinctive movement of WPD-loop occurs during the catalysis of phosphotyrosine by protein tyrosine phosphatase 1B (PTP1B). This loop is in the "open" state in apo-form whereas it is catalytically competent in the "closed" state. During the closure of this loop, unique hydrogen bond interactions are formed between different residues of the PTP1B. Present study examines such interactions from the available 118 crystal structures of PTP1B. It gives insights into the five novel hydrogen bonds essentially formed in the "closed" loop structures. Additionally, the study provides distance ranges between the atoms involved in the hydrogen bonds. This information can be used as a geometrical criterion in the characterization of conformational state of the WPD-loop especially in the molecular dynamics simulations.

Nanoporous frameworks exhibiting multiple stimuli responsiveness

NASA Astrophysics Data System (ADS)

Kundu, Pintu K.; Olsen, Gregory L.; Kiss, Vladimir; Klajn, Rafal

2014-04-01

Nanoporous frameworks are polymeric materials built from rigid molecules, which give rise to their nanoporous structures with applications in gas sorption and storage, catalysis and others. Conceptually new applications could emerge, should these beneficial properties be manipulated by external stimuli in a reversible manner. One approach to render nanoporous frameworks responsive to external signals would be to immobilize molecular switches within their nanopores. Although the majority of molecular switches require conformational freedom to isomerize, and switching in the solid state is prohibited, the nanopores may provide enough room for the switches to efficiently isomerize. Here we describe two families of nanoporous materials incorporating the spiropyran molecular switch. These materials exhibit a variety of interesting properties, including reversible photochromism and acidochromism under solvent-free conditions, light-controlled capture and release of metal ions, as well reversible chromism induced by solvation/desolvation.

Using Local States To Drive the Sampling of Global Conformations in Proteins

PubMed Central

2016-01-01

Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design. PMID:26808351

Molecular dynamics simulations of biological membranes and membrane proteins using enhanced conformational sampling algorithms.

PubMed

Mori, Takaharu; Miyashita, Naoyuki; Im, Wonpil; Feig, Michael; Sugita, Yuji

2016-07-01

This paper reviews various enhanced conformational sampling methods and explicit/implicit solvent/membrane models, as well as their recent applications to the exploration of the structure and dynamics of membranes and membrane proteins. Molecular dynamics simulations have become an essential tool to investigate biological problems, and their success relies on proper molecular models together with efficient conformational sampling methods. The implicit representation of solvent/membrane environments is reasonable approximation to the explicit all-atom models, considering the balance between computational cost and simulation accuracy. Implicit models can be easily combined with replica-exchange molecular dynamics methods to explore a wider conformational space of a protein. Other molecular models and enhanced conformational sampling methods are also briefly discussed. As application examples, we introduce recent simulation studies of glycophorin A, phospholamban, amyloid precursor protein, and mixed lipid bilayers and discuss the accuracy and efficiency of each simulation model and method. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Using Local States To Drive the Sampling of Global Conformations in Proteins.

PubMed

Pandini, Alessandro; Fornili, Arianna

2016-03-08

Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design.

A Mediated Moderation Model of Conformative Peer Bullying

ERIC Educational Resources Information Center

Cho, Yoonju; Chung, Ock-Boon

2012-01-01

We investigated the relationship between conformative peer bullying and issues of peer conformity among adolescents. This relationship is examined through the establishment of a mediated moderation model for conformative peer bullying using structural equation modeling in a sample of 391 second-year middle school students in Seoul, South Korea. We…

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

PubMed

Patel, Sunita; Sasidhar, Yellamraju U

2007-10-01

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

High-quality ZnO inverse opals and related heterostructures as photocatalysts produced by atomic layer deposition

NASA Astrophysics Data System (ADS)

Long, Jie; Fu, Ming; Li, Caixia; Sun, Cuifeng; He, Dawei; Wang, Yongsheng

2018-10-01

ZnO with various nanostructures is widely investigated for high photoelectrochemical (PEC) catalysis performances due to its abundant and inert semiconducting properties with elevated electronic mobility and variable morphologies. Because the solar energy conversion efficiencies could possibly be further enhanced by the introduction of nanophotonic structures with larger surface ratios, high-quality ZnO inverse opals (IOs) were achieved by ALD method using O3 as the oxidant. The intrinsic UV emission peaks and PEC currents of ZnO IOs produced by O3 atomic layer deposition (ALD) method were much improved when compared to those made by H2O ALD and electrodeposition. ALD at higher temperatures (240 °C) can further enhance the crystalline quality and PEC performances. The optimal ALD thickness and filling fraction obtained by controlling ALD cycles, as well as the optimal photonic stop band position obtained by colloidal crystals with different sphere diameters were also discussed. It was found that conformally coated samples with TiO2 protection layers by ALD method using titanium tetrachloride as a precursor enhanced the photochemical stability of ZnO IOs. The photocorrosion was further reduced by inserting ALD Al2O3 inside the TiO2 protection layers. Heterostructured photonic crystals with double-layer IO structures with different pore periodicities were also developed for enhancing the PEC performances.

How accurately do force fields represent protein side chain ensembles?

PubMed

Petrović, Dušan; Wang, Xue; Strodel, Birgit

2018-05-23

Although the protein backbone is the most fundamental part of the structure, the fine-tuning of side-chain conformations is important for protein function, for example, in protein-protein and protein-ligand interactions, and also in enzyme catalysis. While several benchmarks testing the performance of protein force fields for side chain properties have already been published, they often considered only a few force fields and were not tested against the same experimental observables; hence, they are not directly comparable. In this work, we explore the ability of twelve force fields, which are different flavors of AMBER, CHARMM, OPLS, or GROMOS, to reproduce average rotamer angles and rotamer populations obtained from extensive NMR studies of the 3 J and residual dipolar coupling constants for two small proteins: ubiquitin and GB3. Based on a total of 196 μs sampling time, our results reveal that all force fields identify the correct side chain angles, while the AMBER and CHARMM force fields clearly outperform the OPLS and GROMOS force fields in estimating rotamer populations. The three best force fields for representing the protein side chain dynamics are AMBER 14SB, AMBER 99SB*-ILDN, and CHARMM36. Furthermore, we observe that the side chain ensembles of buried amino acid residues are generally more accurately represented than those of the surface exposed residues. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Synthetic, switchable enzymes

PubMed Central

Norris, Vic; Krylov, Sergey N.; Agarwal, Pratul K.; White, Glenn J.

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes alias swenzymes is, in principle, feasible. We propose a strategy to make such catalysts from two (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a two-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker so bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low intensity, non-ionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate, product-capturing, aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. PMID:28448969

Synthetic, Switchable Enzymes.

PubMed

Norris, Vic; Krylov, Sergey N; Agarwal, Pratul K; White, Glenn J

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. © 2017 S. Karger AG, Basel.

Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aleshin,A.; Shiryaev, S.; Strongin, A.

2007-01-01

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis andmore » allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.« less

Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

PubMed Central

Blood, Arlin B.; Tiso, Mauro; Verma, Shilpa T.; Lo, Jennifer; Joshi, Mahesh S.; Azarov, Ivan; Longo, Lawrence D.; Gladwin, Mark T.; Kim-Shapiro, Daniel B.; Power, Gordon G.

2009-01-01

Growing evidence indicates that nitrite, NO2−, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50–80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus. PMID:19028797

Structure of N-acetyl-[beta]-D-glucosaminidase (GcnA) from the Endocarditis Pathogen Streptococcus gordonii and its Complex with the Mechanism-based Inhibitor NAG-thiazoline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Langley, David B.; Harty, Derek W.S.; Jacques, Nicholas A.

2008-09-17

The crystal structure of GcnA, an N-acetyl-{beta}-D-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal {alpha}-helical domain has not been observed previously and forms a large dimerization interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical ({beta}/{alpha}){sub 8} TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a familymore » 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-{beta}-D-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.« less

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism*

PubMed Central

Murray, David S.; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V.; Fisher, Susan H.; Schumacher, Maria A.

2013-01-01

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg62, from an adjacent subunit. Notably, Arg62 must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens. PMID:24158439

Photon catalysis acting as noiseless linear amplification and its application in coherence enhancement

NASA Astrophysics Data System (ADS)

Zhang, Shengli; Zhang, Xiangdong

2018-04-01

Photon catalysis is an intriguing quantum mechanical operation during which no photon is added to or subtracted from the relevant optical system. However, we prove that photon catalysis is in essence equivalent to the simpler but more efficient noiseless linear amplifier. This provides a simple and zero-energy-input method for enhancing quantum coherence. We show that the coherence enhancement holds both for a coherent state and a two-mode squeezed vacuum (TMSV) state. For the TMSV state, biside photon catalysis is shown to be equivalent to two times the single-side photon catalysis, and two times the photon catalysis does not provide a substantial enhancement of quantum coherence compared with single-side catalysis. We further extend our investigation to the performance of coherence enhancement with a more realistic photon catalysis scheme where a heralded approximated single-photon state and an on-off detector are exploited. Moreover, we investigate the influence of an imperfect photon detector and the result shows that the amplification effect of photon catalysis is insensitive to the detector inefficiency. Finally, we apply the coherence measure to quantum illumination and see the same trend of performance improvement as coherence enhancement is identified in practical quantum target detection.

Replication infidelity via a mismatch with Watson–Crick geometry

PubMed Central

Bebenek, Katarzyna; Pedersen, Lars C.; Kunkel, Thomas A.

2011-01-01

In describing the DNA double helix, Watson and Crick suggested that “spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms.” Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson–Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base–base mismatch with Watson–Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase λ variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson–Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson–Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G•T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA. PMID:21233421

Replication infidelity via a mismatch with Watson-Crick geometry.

PubMed

Bebenek, Katarzyna; Pedersen, Lars C; Kunkel, Thomas A

2011-02-01

In describing the DNA double helix, Watson and Crick suggested that "spontaneous mutation may be due to a base occasionally occurring in one of its less likely tautomeric forms." Indeed, among many mispairing possibilities, either tautomerization or ionization of bases might allow a DNA polymerase to insert a mismatch with correct Watson-Crick geometry. However, despite substantial progress in understanding the structural basis of error prevention during polymerization, no DNA polymerase has yet been shown to form a natural base-base mismatch with Watson-Crick-like geometry. Here we provide such evidence, in the form of a crystal structure of a human DNA polymerase λ variant poised to misinsert dGTP opposite a template T. All atoms needed for catalysis are present at the active site and in positions that overlay with those for a correct base pair. The mismatch has Watson-Crick geometry consistent with a tautomeric or ionized base pair, with the pH dependence of misinsertion consistent with the latter. The results support the original idea that a base substitution can originate from a mismatch having Watson-Crick geometry, and they suggest a common catalytic mechanism for inserting a correct and an incorrect nucleotide. A second structure indicates that after misinsertion, the now primer-terminal G • T mismatch is also poised for catalysis but in the wobble conformation seen in other studies, indicating the dynamic nature of the pathway required to create a mismatch in fully duplex DNA.

Tackling the conformational sampling of larger flexible compounds and macrocycles in pharmacology and drug discovery.

PubMed

Chen, I-Jen; Foloppe, Nicolas

2013-12-15

Computational conformational sampling underpins much of molecular modeling and design in pharmaceutical work. The sampling of smaller drug-like compounds has been an active area of research. However, few studies have tested in details the sampling of larger more flexible compounds, which are also relevant to drug discovery, including therapeutic peptides, macrocycles, and inhibitors of protein-protein interactions. Here, we investigate extensively mainstream conformational sampling methods on three carefully curated compound sets, namely the 'Drug-like', larger 'Flexible', and 'Macrocycle' compounds. These test molecules are chemically diverse with reliable X-ray protein-bound bioactive structures. The compared sampling methods include Stochastic Search and the recent LowModeMD from MOE, all the low-mode based approaches from MacroModel, and MD/LLMOD recently developed for macrocycles. In addition to default settings, key parameters of the sampling protocols were explored. The performance of the computational protocols was assessed via (i) the reproduction of the X-ray bioactive structures, (ii) the size, coverage and diversity of the output conformational ensembles, (iii) the compactness/extendedness of the conformers, and (iv) the ability to locate the global energy minimum. The influence of the stochastic nature of the searches on the results was also examined. Much better results were obtained by adopting search parameters enhanced over the default settings, while maintaining computational tractability. In MOE, the recent LowModeMD emerged as the method of choice. Mixed torsional/low-mode from MacroModel performed as well as LowModeMD, and MD/LLMOD performed well for macrocycles. The low-mode based approaches yielded very encouraging results with the flexible and macrocycle sets. Thus, one can productively tackle the computational conformational search of larger flexible compounds for drug discovery, including macrocycles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors.

PubMed

Zhang, Yajia; Hauser, Kris

2013-01-01

Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion.

Unbiased, scalable sampling of protein loop conformations from probabilistic priors

PubMed Central

2013-01-01

Background Protein loops are flexible structures that are intimately tied to function, but understanding loop motion and generating loop conformation ensembles remain significant computational challenges. Discrete search techniques scale poorly to large loops, optimization and molecular dynamics techniques are prone to local minima, and inverse kinematics techniques can only incorporate structural preferences in adhoc fashion. This paper presents Sub-Loop Inverse Kinematics Monte Carlo (SLIKMC), a new Markov chain Monte Carlo algorithm for generating conformations of closed loops according to experimentally available, heterogeneous structural preferences. Results Our simulation experiments demonstrate that the method computes high-scoring conformations of large loops (>10 residues) orders of magnitude faster than standard Monte Carlo and discrete search techniques. Two new developments contribute to the scalability of the new method. First, structural preferences are specified via a probabilistic graphical model (PGM) that links conformation variables, spatial variables (e.g., atom positions), constraints and prior information in a unified framework. The method uses a sparse PGM that exploits locality of interactions between atoms and residues. Second, a novel method for sampling sub-loops is developed to generate statistically unbiased samples of probability densities restricted by loop-closure constraints. Conclusion Numerical experiments confirm that SLIKMC generates conformation ensembles that are statistically consistent with specified structural preferences. Protein conformations with 100+ residues are sampled on standard PC hardware in seconds. Application to proteins involved in ion-binding demonstrate its potential as a tool for loop ensemble generation and missing structure completion. PMID:24565175

Latent Class Analysis of Peer Conformity: Who Is Yielding to Pressure and Why?

ERIC Educational Resources Information Center

Kosten, Paul A.; Scheier, Lawrence M.; Grenard, Jerry L.

2013-01-01

This study used latent class analysis to examine typologies of peer conformity in a community sample of middle school students. Students responded to 31 items assessing diverse facets of conformity dispositions. The most parsimonious model produced three qualitatively distinct classes that differed on the basis of conformity to recreational…

LeuT conformational sampling utilizing accelerated molecular dynamics and principal component analysis.

PubMed

Thomas, James R; Gedeon, Patrick C; Grant, Barry J; Madura, Jeffry D

2012-07-03

Monoamine transporters (MATs) function by coupling ion gradients to the transport of dopamine, norepinephrine, or serotonin. Despite their importance in regulating neurotransmission, the exact conformational mechanism by which MATs function remains elusive. To this end, we have performed seven 250 ns accelerated molecular dynamics simulations of the leucine transporter, a model for neurotransmitter MATs. By varying the presence of binding-pocket leucine substrate and sodium ions, we have sampled plausible conformational states representative of the substrate transport cycle. The resulting trajectories were analyzed using principal component analysis of transmembrane helices 1b and 6a. This analysis revealed seven unique structures: two of the obtained conformations are similar to the currently published crystallographic structures, one conformation is similar to a proposed open inward structure, and four conformations represent novel structures of potential importance to the transport cycle. Further analysis reveals that the presence of binding-pocket sodium ions is necessary to stabilize the locked-occluded and open-inward conformations. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

[Non-conformities management in laboratory of medical biology: application to non-conformities of biological samples during 2009].

PubMed

Annaix, Véronique; Rogowski, Julien; Joyau, Mireille; Jaouën, Edtih

2011-01-01

The non-conformity management is required for the ISO 15189 standard. The laboratory of medical biology has to carry out suitable acts and procedures to exploit different indicators through the framework of continuous improvement. We particularly study the indicator of biological samples nonconformities and we report 2009 results to the nurses' team managers to find solutions for quality of care to the patient.

Characterizing Conformational Dynamics of Proteins Using Evolutionary Couplings.

PubMed

Feng, Jiangyan; Shukla, Diwakar

2018-01-25

Understanding of protein conformational dynamics is essential for elucidating molecular origins of protein structure-function relationship. Traditionally, reaction coordinates, i.e., some functions of protein atom positions and velocities have been used to interpret the complex dynamics of proteins obtained from experimental and computational approaches such as molecular dynamics simulations. However, it is nontrivial to identify the reaction coordinates a priori even for small proteins. Here, we evaluate the power of evolutionary couplings (ECs) to capture protein dynamics by exploring their use as reaction coordinates, which can efficiently guide the sampling of a conformational free energy landscape. We have analyzed 10 diverse proteins and shown that a few ECs are sufficient to characterize complex conformational dynamics of proteins involved in folding and conformational change processes. With the rapid strides in sequencing technology, we expect that ECs could help identify reaction coordinates a priori and enhance the sampling of the slow dynamical process associated with protein folding and conformational change.

Achieving Rigorous Accelerated Conformational Sampling in Explicit Solvent.

PubMed

Doshi, Urmi; Hamelberg, Donald

2014-04-03

Molecular dynamics simulations can provide valuable atomistic insights into biomolecular function. However, the accuracy of molecular simulations on general-purpose computers depends on the time scale of the events of interest. Advanced simulation methods, such as accelerated molecular dynamics, have shown tremendous promise in sampling the conformational dynamics of biomolecules, where standard molecular dynamics simulations are nonergodic. Here we present a sampling method based on accelerated molecular dynamics in which rotatable dihedral angles and nonbonded interactions are boosted separately. This method (RaMD-db) is a different implementation of the dual-boost accelerated molecular dynamics, introduced earlier. The advantage is that this method speeds up sampling of the conformational space of biomolecules in explicit solvent, as the degrees of freedom most relevant for conformational transitions are accelerated. We tested RaMD-db on one of the most difficult sampling problems - protein folding. Starting from fully extended polypeptide chains, two fast folding α-helical proteins (Trpcage and the double mutant of C-terminal fragment of Villin headpiece) and a designed β-hairpin (Chignolin) were completely folded to their native structures in very short simulation time. Multiple folding/unfolding transitions could be observed in a single trajectory. Our results show that RaMD-db is a promisingly fast and efficient sampling method for conformational transitions in explicit solvent. RaMD-db thus opens new avenues for understanding biomolecular self-assembly and functional dynamics occurring on long time and length scales.

Conformational properties of chiral tobacco alkaloids by DFT calculations and vibrational circular dichroism: (-)-S-anabasine.

PubMed

Rodríguez Ortega, P G; Montejo, M; Márquez, F; López González, J J

2015-07-01

A thorough DFT and MM study of the conformational landscape, molecular and electronic structures of (-)-S-anabasine is reported aimed to reveal the mechanism controlling its conformational preference. Although the conformational flexibility and diversity of this system is quite extensive, only two structures are populated both in gas-phase and solution (CCl4 and DMSO). NBO-aided electronic structure analyses performed for the eight conformers representing minima in the potential energy surface of (-)-S-anabasine indicate that both steric and electrostatic factors are determinant in the conformational distribution of the sample in gas phase. Nonetheless, hyperconjugative effects are the key force tipping the balance in the conformational equilibrium between the two main rotamers. Increasing the polarity of the medium (using the IEF-PCM formalism) barely affect the conformational energy profile, although a slight increase in the theoretical population of those structures more affected by electrostatic interactions is predicted. The validity of the theoretical models and calculated conformers populations are endorsed by the accurate reproduction of the IR and VCD spectra (recorded in pure liquid and in CCl4 solution) of the sample (that have been firstly recorded and assigned in the present work) which are consistent with the occurrence of a 2:1 conformational ratio. Copyright © 2015 Elsevier Inc. All rights reserved.

Enhanced Conformational Sampling of N-Glycans in Solution with Replica State Exchange Metadynamics.

PubMed

Galvelis, Raimondas; Re, Suyong; Sugita, Yuji

2017-05-09

Molecular dynamics (MD) simulation of a N-glycan in solution is challenging because of high-energy barriers of the glycosidic linkages, functional group rotational barriers, and numerous intra- and intermolecular hydrogen bonds. In this study, we apply different enhanced conformational sampling approaches, namely, metadynamics (MTD), the replica-exchange MD (REMD), and the recently proposed replica state exchange MTD (RSE-MTD), to a N-glycan in solution and compare the conformational sampling efficiencies of the approaches. MTD helps to cross the high-energy barrier along the ω angle by utilizing a bias potential, but it cannot enhance sampling of the other degrees of freedom. REMD ensures moderate-energy barrier crossings by exchanging temperatures between replicas, while it hardly crosses the barriers along ω. In contrast, RSE-MTD succeeds to cross the high-energy barrier along ω as well as to enhance sampling of the other degrees of freedom. We tested two RSE-MTD schemes: in one scheme, 64 replicas were simulated with the bias potential along ω at different temperatures, while simulations of four replicas were performed with the bias potentials for different CVs at 300 K. In both schemes, one unbiased replica at 300 K was included to compute conformational properties of the glycan. The conformational sampling of the former is better than the other enhanced sampling methods, while the latter shows reasonable performance without spending large computational resources. The latter scheme is likely to be useful when a N-glycan-attached protein is simulated.

Enhanced Conformational Sampling in Molecular Dynamics Simulations of Solvated Peptides: Fragment-Based Local Elevation Umbrella Sampling.

PubMed

Hansen, Halvor S; Daura, Xavier; Hünenberger, Philippe H

2010-09-14

A new method, fragment-based local elevation umbrella sampling (FB-LEUS), is proposed to enhance the conformational sampling in explicit-solvent molecular dynamics (MD) simulations of solvated polymers. The method is derived from the local elevation umbrella sampling (LEUS) method [ Hansen and Hünenberger , J. Comput. Chem. 2010 , 31 , 1 - 23 ], which combines the local elevation (LE) conformational searching and the umbrella sampling (US) conformational sampling approaches into a single scheme. In LEUS, an initial (relatively short) LE build-up (searching) phase is used to construct an optimized (grid-based) biasing potential within a subspace of conformationally relevant degrees of freedom, which is then frozen and used in a (comparatively longer) US sampling phase. This combination dramatically enhances the sampling power of MD simulations but, due to computational and memory costs, is only applicable to relevant subspaces of low dimensionalities. As an attempt to expand the scope of the LEUS approach to solvated polymers with more than a few relevant degrees of freedom, the FB-LEUS scheme involves an US sampling phase that relies on a superposition of low-dimensionality biasing potentials optimized using LEUS at the fragment level. The feasibility of this approach is tested using polyalanine (poly-Ala) and polyvaline (poly-Val) oligopeptides. Two-dimensional biasing potentials are preoptimized at the monopeptide level, and subsequently applied to all dihedral-angle pairs within oligopeptides of 4,  6,  8, or 10 residues. Two types of fragment-based biasing potentials are distinguished: (i) the basin-filling (BF) potentials act so as to "fill" free-energy basins up to a prescribed free-energy level above the global minimum; (ii) the valley-digging (VD) potentials act so as to "dig" valleys between the (four) free-energy minima of the two-dimensional maps, preserving barriers (relative to linearly interpolated free-energy changes) of a prescribed magnitude. The application of these biasing potentials may lead to an impressive enhancement of the searching power (volume of conformational space visited in a given amount of simulation time). However, this increase is largely offset by a deterioration of the statistical efficiency (representativeness of the biased ensemble in terms of the conformational distribution appropriate for the physical ensemble). As a result, it appears difficult to engineer FB-LEUS schemes representing a significant improvement over plain MD, at least for the systems considered here.

A population-based evolutionary search approach to the multiple minima problem in de novo protein structure prediction

PubMed Central

2013-01-01

Background Elucidating the native structure of a protein molecule from its sequence of amino acids, a problem known as de novo structure prediction, is a long standing challenge in computational structural biology. Difficulties in silico arise due to the high dimensionality of the protein conformational space and the ruggedness of the associated energy surface. The issue of multiple minima is a particularly troublesome hallmark of energy surfaces probed with current energy functions. In contrast to the true energy surface, these surfaces are weakly-funneled and rich in comparably deep minima populated by non-native structures. For this reason, many algorithms seek to be inclusive and obtain a broad view of the low-energy regions through an ensemble of low-energy (decoy) conformations. Conformational diversity in this ensemble is key to increasing the likelihood that the native structure has been captured. Methods We propose an evolutionary search approach to address the multiple-minima problem in decoy sampling for de novo structure prediction. Two population-based evolutionary search algorithms are presented that follow the basic approach of treating conformations as individuals in an evolving population. Coarse graining and molecular fragment replacement are used to efficiently obtain protein-like child conformations from parents. Potential energy is used both to bias parent selection and determine which subset of parents and children will be retained in the evolving population. The effect on the decoy ensemble of sampling minima directly is measured by additionally mapping a conformation to its nearest local minimum before considering it for retainment. The resulting memetic algorithm thus evolves not just a population of conformations but a population of local minima. Results and conclusions Results show that both algorithms are effective in terms of sampling conformations in proximity of the known native structure. The additional minimization is shown to be key to enhancing sampling capability and obtaining a diverse ensemble of decoy conformations, circumventing premature convergence to sub-optimal regions in the conformational space, and approaching the native structure with proximity that is comparable to state-of-the-art decoy sampling methods. The results are shown to be robust and valid when using two representative state-of-the-art coarse-grained energy functions. PMID:24565020

Exploiting enzyme catalysis in ultra-low ion strength media for impedance biosensing of avian influenza virus using a bare interdigitated electrode.

PubMed

Fu, Yingchun; Callaway, Zachary; Lum, Jacob; Wang, Ronghui; Lin, Jianhan; Li, Yanbin

2014-02-18

Enzyme catalysis is broadly used in various fields but generally applied in media with high ion strength. Here, we propose the exploitation of enzymatic catalysis in ultra-low ion strength media to induce ion strength increase for developing a novel impedance biosensing method. Avian influenza virus H5N1, a serious worldwide threat to poultry and human health, was adopted as the analyte. Magnetic beads were modified with H5N1-specific aptamer to capture the H5N1 virus. This was followed by binding concanavalin A (ConA), glucose oxidase (GOx), and Au nanoparticles (AuNPs) to create bionanocomposites through a ConA-glycan interaction. The yielded sandwich complex was transferred to a glucose solution to trigger an enzymatic reaction to produce gluconic acid, which ionized to increase the ion strength of the solution, thus decreasing the impedance on a screen-printed interdigitated array electrode. This method took advantages of the high efficiency of enzymatic catalysis and the high susceptibility of electrochemical impedance on the ion strength and endowed the biosensor with high sensitivity and a detection limit of 8 × 10(-4) HAU in 200 μL sample, which was magnitudes lower than that of some analogues based on biosensing methods. Furthermore, the proposed method required only a bare electrode for measurements of ion strength change and had negligible change on the surficial properties of the electrode, though some modification of magnetic beads/Au nanoparticles and the construction of a sandwich complex were still needed. This helped to avoid the drawbacks of commonly used electrode immobilization methods. The merit for this method makes it highly useful and promising for applications. The proposed method may create new possibilities in the broad and well-developed enzymatic catalysis fields and find applications in developing sensitive, rapid, low-cost, and easy-to-operate biosensing and biocatalysis devices.

Synergistic Catalysis: A Powerful Synthetic Strategy for New Reaction Development

PubMed Central

Allen, Anna E.; MacMillan, David W. C.

2012-01-01

Synergistic catalysis is a synthetic strategy wherein both the nucleophile and the electrophile are simultaneously activated by two separate and distinct catalysts to afford a single chemical transformation. This powerful catalysis strategy leads to several benefits, specifically synergistic catalysis can (i) introduce new, previously unattainable chemical transformations, (ii) improve the efficiency of existing transformations, and (iii) create or improve catalytic enantioselectivity where stereocontrol was previously absent or challenging. This perspective aims to highlight these benefits using many of the successful examples of synergistic catalysis found in the literature. PMID:22518271

Conformational Heterogeneity of Unbound Proteins Enhances Recognition in Protein-Protein Encounters.

PubMed

Pallara, Chiara; Rueda, Manuel; Abagyan, Ruben; Fernández-Recio, Juan

2016-07-12

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.

Synergistic amylomaltase and branching enzyme catalysis to suppress cassava starch digestibility.

PubMed

Sorndech, Waraporn; Meier, Sebastian; Jansson, Anita M; Sagnelli, Domenico; Hindsgaul, Ole; Tongta, Sunanta; Blennow, Andreas

2015-11-05

Starch provides our main dietary caloric intake and over-consumption of starch-containing foods results in escalating life-style disease including diabetes. By increasing the content of α-1,6 branch points in starch, digestibility by human amylolytic enzymes is expected to be retarded. Aiming at generating a soluble and slowly digestible starch by increasing the content and changing the relative positioning of the branch points in the starch molecules, we treated cassava starch with amylomaltase (AM) and branching enzyme (BE). We performed a detailed molecular analysis of the products including amylopectin chain length distribution, content of α-1,6 glucosidic linkages, absolute molecular weight distribution and digestibility. Step-by-step enzyme catalysis was the most efficient treatment, and it generated branch structures even more extreme than those of glycogen. All AM- and BE-treated samples showed increased resistance to degradation by porcine pancreatic α-amylase and glucoamylase as compared to cassava starch. The amylolytic products showed chain lengths and branching patterns similar to the products obtained from glycogen. Our data demonstrate that combinatorial enzyme catalysis provides a strategy to generate potential novel soluble α-glucan ingredients with low dietary digestibility assets. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vapor deposition routes to conformal polymer thin films

PubMed Central

Moni, Priya; Al-Obeidi, Ahmed

2017-01-01

Vapor phase syntheses, including parylene chemical vapor deposition (CVD) and initiated CVD, enable the deposition of conformal polymer thin films to benefit a diverse array of applications. This short review for nanotechnologists, including those new to vapor deposition methods, covers the basic theory in designing a conformal polymer film vapor deposition, sample preparation and imaging techniques to assess film conformality, and several applications that have benefited from vapor deposited, conformal polymer thin films. PMID:28487816

Force-momentum-based self-guided Langevin dynamics: A rapid sampling method that approaches the canonical ensemble

NASA Astrophysics Data System (ADS)

Wu, Xiongwu; Brooks, Bernard R.

2011-11-01

The self-guided Langevin dynamics (SGLD) is a method to accelerate conformational searching. This method is unique in the way that it selectively enhances and suppresses molecular motions based on their frequency to accelerate conformational searching without modifying energy surfaces or raising temperatures. It has been applied to studies of many long time scale events, such as protein folding. Recent progress in the understanding of the conformational distribution in SGLD simulations makes SGLD also an accurate method for quantitative studies. The SGLD partition function provides a way to convert the SGLD conformational distribution to the canonical ensemble distribution and to calculate ensemble average properties through reweighting. Based on the SGLD partition function, this work presents a force-momentum-based self-guided Langevin dynamics (SGLDfp) simulation method to directly sample the canonical ensemble. This method includes interaction forces in its guiding force to compensate the perturbation caused by the momentum-based guiding force so that it can approximately sample the canonical ensemble. Using several example systems, we demonstrate that SGLDfp simulations can approximately maintain the canonical ensemble distribution and significantly accelerate conformational searching. With optimal parameters, SGLDfp and SGLD simulations can cross energy barriers of more than 15 kT and 20 kT, respectively, at similar rates for LD simulations to cross energy barriers of 10 kT. The SGLDfp method is size extensive and works well for large systems. For studies where preserving accessible conformational space is critical, such as free energy calculations and protein folding studies, SGLDfp is an efficient approach to search and sample the conformational space.

Homogeneous, Heterogeneous, and Enzymatic Catalysis.

ERIC Educational Resources Information Center

Oyama, S. Ted; Somorjai, Gabor A.

1988-01-01

Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

Enantioselective conjugate additions of α-amino radicals via cooperative photoredox and Lewis acid catalysis.

PubMed

Ruiz Espelt, Laura; McPherson, Iain S; Wiensch, Eric M; Yoon, Tehshik P

2015-02-25

We report the highly enantioselective addition of photogenerated α-amino radicals to Michael acceptors. This method features a dual-catalyst protocol that combines transition metal photoredox catalysis with chiral Lewis acid catalysis. The combination of these two powerful modes of catalysis provides an effective, general strategy to generate and control the reactivity of photogenerated reactive intermediates.

Fast de novo discovery of low-energy protein loop conformations.

PubMed

Wong, Samuel W K; Liu, Jun S; Kou, S C

2017-08-01

In the prediction of protein structure from amino acid sequence, loops are challenging regions for computational methods. Since loops are often located on the protein surface, they can have significant roles in determining protein functions and binding properties. Loop prediction without the aid of a structural template requires extensive conformational sampling and energy minimization, which are computationally difficult. In this article we present a new de novo loop sampling method, the Parallely filtered Energy Targeted All-atom Loop Sampler (PETALS) to rapidly locate low energy conformations. PETALS explores both backbone and side-chain positions of the loop region simultaneously according to the energy function selected by the user, and constructs a nonredundant ensemble of low energy loop conformations using filtering criteria. The method is illustrated with the DFIRE potential and DiSGro energy function for loops, and shown to be highly effective at discovering conformations with near-native (or better) energy. Using the same energy function as the DiSGro algorithm, PETALS samples conformations with both lower RMSDs and lower energies. PETALS is also useful for assessing the accuracy of different energy functions. PETALS runs rapidly, requiring an average time cost of 10 minutes for a length 12 loop on a single 3.2 GHz processor core, comparable to the fastest existing de novo methods for generating an ensemble of conformations. Proteins 2017; 85:1402-1412. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Nanoparticle-based biologic mimetics

PubMed Central

Cliffel, David E.; Turner, Brian N.; Huffman, Brian J.

2009-01-01

Centered on solid chemistry foundations, biology and materials science have reached a crossroad where bottom-up designs of new biologically important nanomaterials are a reality. The topics discussed here present the interdisciplinary field of creating biological mimics. Specifically, this discussion focuses on mimics that are developed using various types of metal nanoparticles (particularly gold) through facile synthetic methods. These methods conjugate biologically relevant molecules, e.g., small molecules, peptides, proteins, and carbohydrates, in conformationally favorable orientations on the particle surface. These new products provide stable, safe, and effective substitutes for working with potentially hazardous biologicals for applications such as drug targeting, immunological studies, biosensor development, and biocatalysis. Many standard bioanalytical techniques can be used to characterize and validate the efficacy of these new materials, including quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). Metal nanoparticle–based biomimetics continue to be developed as potential replacements for the native biomolecule in applications of immunoassays and catalysis. PMID:20049778

Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Westfall, Corey S.; Zubieta, Chloe; Herrmann, Jonathan

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how amore » highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.« less

Approaches to efficiently estimate solvation and explicit water energetics in ligand binding: the use of WaterMap [WaterMap and Its Implementation in Drug Discovery

DOE PAGES

Yang, Yue; Wong, Sergio E.; Lightstone, Felice C.

2012-09-08

Solvents play quite an important role in most chemical and biological processes. It is widely accepted that the presence of water or other solvents in many chemical reactions can result in much lower energy barrier. In enzymatic catalysis, water mediated reaction pathways have been observed in various studies. In addition, different conformation flexibility and hydrogen bond patterns have been discovered for cyclic peptides in the presence of membrane and water, further illustrating the impact of solvent in biological activities such like membrane penetration. moreover, as will be discussed later in this review, water also plays a critical role in host-guestmore » chemistry and thus is essential to drug design. As such, it is not surprising that accounting for solvents is critical in drug discovery since drugs must modulate biological systems.« less

Origins of the temperature dependence of hammerhead ribozyme catalysis.

PubMed Central

Peracchi, A

1999-01-01

The difficulties in interpreting the temperature dependence of protein enzyme reactions are well recognized. Here, the hammerhead ribozyme cleavage was investigated under single-turnover conditions between 0 and 60 degrees C as a model for RNA-catalyzed reactions. Under the adopted conditions, the chemical step appears to be rate-limiting. However, the observed rate of cleavage is affected by pre-catalytic equilibria involving deprotonation of an essential group and binding of at least one low-affinity Mg2+ion. Thus, the apparent entropy and enthalpy of activation include contributions from the temperature dependence of these equilibria, precluding a simple physical interpretation of the observed activation parameters. Similar pre-catalytic equilibria likely contribute to the observed activation parameters for ribozyme reactions in general. The Arrhenius plot for the hammerhead reaction is substantially curved over the temperature range considered, which suggests the occurrence of a conformational change of the ribozyme ground state around physiological temperatures. PMID:10390528

Recent advances in the applications of ionic liquids in protein stability and activity: a review.

PubMed

Patel, Rajan; Kumari, Meena; Khan, Abbul Bashar

2014-04-01

Room temperatures ionic liquids are considered as miraculous solvents for biological system. Due to their inimitable properties and large variety of applications, they have been widely used in enzyme catalysis and protein stability and separation. The related information present in the current review is helpful to the researchers working in the field of biotechnology and biochemistry to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. We have extensively analyzed the methods used for studying the protein-IL interaction which is useful in providing information about structural and conformational dynamics of protein. This can be helpful to develop and understanding about the effect of ionic liquids on stability and activity of proteins. In addition, the affect of physico-chemical properties of ionic liquids, viz. hydrogen bond capacity and hydrophobicity on protein stability are discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Xiangyu; Qin, Xiangjing; Chen, Lei

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunitmore » fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).« less

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

PubMed

Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-06-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

PubMed Central

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. PMID:23559560

Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue†‡

PubMed Central

Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika; Sanishvili, Ruslan; Xu, Chengfu; Viola, Ronald E.

2009-01-01

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-l-aspartate to produce l-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-l-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-l-aspartate. PMID:18293939

Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue.

PubMed

Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika; Sanishvili, Ruslan; Xu, Chengfu; Viola, Ronald E

2008-03-18

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.

High-Dimensional Mutant and Modular Thermodynamic Cycles, Molecular Switching, and Free Energy Transduction

PubMed Central

Carter, Charles W.

2017-01-01

Understanding how distinct parts of proteins produce coordinated behavior has driven and continues to drive advances in protein science and enzymology. However, despite consensus about the conceptual basis for allostery, the idiosyncratic nature of allosteric mechanisms resists general approaches. Computational methods can identify conformational transition states from structural changes, revealing common switching mechanisms that impose multistate behavior. Thermodynamic cycles use factorial perturbations to measure coupling energies between side chains in molecular switches that mediate shear during domain motion. Such cycles have now been complemented by modular cycles that measure energetic coupling between separable domains. For one model system, energetic coupling between domains has been shown to be quantitatively equivalent to that between dynamic side chains. Linkages between domain motion, switching residues, and catalysis make nucleoside triphosphate hydrolysis conditional on domain movement, confirming an essential yet neglected aspect of free energy transduction and suggesting the potential generality of these studies. PMID:28375734

A Hard X-ray Study of a Manganese-Terpyridine Dimer Catalyst in a Chromium-based Metal Organic Framework - Oral Presentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramsey, Alexandra

2015-08-25

Cleaner forms of energy are needed, and H 2 produced from water spliFng is a possible source. However, a robust catalyst is necessary to carry out the water oxidaKon reacKon. Plants uKlize Photosystem II to catalyze water oxidaKon as a part of photosynthesis, and many syntheKc water oxidaKon catalysts use Photosystem II as a model. In this study, the catalyst of interest was [(terpy)Mn(μ-O)2Mn(terpy)]3+ (MnTD), which was synthesized in a chromium-based Metal Organic Framework (MOF) to avoid degradaKon of MnTD molecules. Hard X-ray powder diffracKon was the primary method of analysis. The diffracKon data was used to detect the presencemore » of MOF in samples at different catalyKc stages, and laFce parameters were assigned to the samples containing MOF. Fourier maps were constructed to determine the contents of the MOF as preliminary studies suggested that MnTD may not be present. Results showed that MOF is present before catalysis occurs, but disappears in the iniKal stages of catalysis. Changes in the MOF’s laFce parameters suggest aWracKve interacKons between the MOF and catalyst; these interacKons may lead to the observed MOF degradaKon. Fourier maps also reveal limited, if any, amounts of MnTD in the system. Molecular manganese oxide may be the source of the high rate of water oxidaKon catalysis in the studied system.« less

Fluctuation Flooding Method (FFM) for accelerating conformational transitions of proteins.

PubMed

Harada, Ryuhei; Takano, Yu; Shigeta, Yasuteru

2014-03-28

A powerful conformational sampling method for accelerating structural transitions of proteins, "Fluctuation Flooding Method (FFM)," is proposed. In FFM, cycles of the following steps enhance the transitions: (i) extractions of largely fluctuating snapshots along anisotropic modes obtained from trajectories of multiple independent molecular dynamics (MD) simulations and (ii) conformational re-sampling of the snapshots via re-generations of initial velocities when re-starting MD simulations. In an application to bacteriophage T4 lysozyme, FFM successfully accelerated the open-closed transition with the 6 ns simulation starting solely from the open state, although the 1-μs canonical MD simulation failed to sample such a rare event.

Fluctuation Flooding Method (FFM) for accelerating conformational transitions of proteins

NASA Astrophysics Data System (ADS)

Harada, Ryuhei; Takano, Yu; Shigeta, Yasuteru

2014-03-01

A powerful conformational sampling method for accelerating structural transitions of proteins, "Fluctuation Flooding Method (FFM)," is proposed. In FFM, cycles of the following steps enhance the transitions: (i) extractions of largely fluctuating snapshots along anisotropic modes obtained from trajectories of multiple independent molecular dynamics (MD) simulations and (ii) conformational re-sampling of the snapshots via re-generations of initial velocities when re-starting MD simulations. In an application to bacteriophage T4 lysozyme, FFM successfully accelerated the open-closed transition with the 6 ns simulation starting solely from the open state, although the 1-μs canonical MD simulation failed to sample such a rare event.

Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallen,J.; Paige, C.; Mallett, T.

2008-01-01

We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

Dynamics differentiate between active and inactive inteins

PubMed Central

Cronin, Melissa; Coolbaugh, Michael J; Nellis, David; Zhu, Jianwei; Wood, David W.; Nussinov, Ruth; Ma, Buyong

2014-01-01

The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving. PMID:25087201

Heterosubunit composition and crystal structures of a novel bacterial M16B metallopeptidase.

PubMed

Maruyama, Yukie; Chuma, Asako; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

2011-03-18

Three subfamilies of metallopeptidase family M16 enzymes--M16A, M16B, and M16C--are widely distributed among eukaryotes and prokaryotes. SPH2681, a periplasmic M16B protein found in Sphingomonas sp. strain A1, contains an HXXEH motif essential for Zn(2+) binding and catalytic activity. SPH2682 is another member of M16B, which lacks the metal-binding motif but conserves an active-site R/Y pair commonly found in the C-terminal half of M16 enzymes. Two genes coding for SPH2681 and SPH2682 assemble into a single operon in the bacterial genome. This study determined SPH2681 to be constitutively expressed in strain A1 cells grown on different carbon sources, suggesting a more general cellular function. SPH2681 and SPH2681/SPH2682 were overexpressed in Escherichia coli, purified, and characterized. SPH2681 was found to associate with SPH2682, forming a heterosubunit enzyme with peptidase activity, while SPH2681 alone exhibited no enzymatic activity. X-ray crystallography of the SPH2681/SPH2682 complex revealed two conformations (open and closed heterodimeric forms) within the same crystal. Compared with the closed form, the open form contains two subunits rotated away from each other by approximately 8°, increasing the distance between the zinc ion and active-site residues by up to 8 Å. In addition, many hydrogen bonds are formed or broken on change between the conformations of the heterodimers, suggesting that subunit dynamics is a prerequisite for catalysis. To our knowledge, this is the first report on both conformational forms of the same M16 peptidase, providing a unique insight into the general proteolytic mechanism of M16 proteases. Copyright © 2011 Elsevier Ltd. All rights reserved.

On the mechanism of peptidoglycan binding and cleavage by the endo-specific lytic transglycosylase MltE from Escherichia coli.

PubMed

Fibriansah, Guntur; Gliubich, Francesca I; Thunnissen, Andy-Mark W H

2012-11-13

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites -4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites -1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a (4)C(1) chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite -1 and by anchimeric assistance of the sugar's N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

PubMed Central

Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

2011-01-01

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

PubMed

Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

2017-07-21

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Enantioselective Conjugate Additions of α-Amino Radicals via Cooperative Photoredox and Lewis Acid Catalysis

PubMed Central

Espelt, Laura Ruiz; McPherson, Iain S.; Wiensch, Eric M.; Yoon, Tehshik P.

2015-01-01

We report the highly enantioselective addition of photogenerated α-amino radicals to Michael acceptors. This method features a dual-catalyst protocol that combines transition metal photoredox catalysis with chiral Lewis acid catalysis. The combination of these two powerful modes of catalysis provides an effective, general strategy to generate and control the reactivity of photogenerated reactive intermediates. PMID:25668687

Seventh BES (Basic Energy Sciences) catalysis and surface chemistry research conference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1990-03-01

Research programs on catalysis and surface chemistry are presented. A total of fifty-seven topics are included. Areas of research include heterogeneous catalysis; catalysis in hydrogenation, desulfurization, gasification, and redox reactions; studies of surface properties and surface active sites; catalyst supports; chemical activation, deactivation; selectivity, chemical preparation; molecular structure studies; sorption and dissociation. Individual projects are processed separately for the data bases. (CBS)

Accelerated Adaptive Integration Method

PubMed Central

2015-01-01

Conformational changes that occur upon ligand binding may be too slow to observe on the time scales routinely accessible using molecular dynamics simulations. The adaptive integration method (AIM) leverages the notion that when a ligand is either fully coupled or decoupled, according to λ, barrier heights may change, making some conformational transitions more accessible at certain λ values. AIM adaptively changes the value of λ in a single simulation so that conformations sampled at one value of λ seed the conformational space sampled at another λ value. Adapting the value of λ throughout a simulation, however, does not resolve issues in sampling when barriers remain high regardless of the λ value. In this work, we introduce a new method, called Accelerated AIM (AcclAIM), in which the potential energy function is flattened at intermediate values of λ, promoting the exploration of conformational space as the ligand is decoupled from its receptor. We show, with both a simple model system (Bromocyclohexane) and the more complex biomolecule Thrombin, that AcclAIM is a promising approach to overcome high barriers in the calculation of free energies, without the need for any statistical reweighting or additional processors. PMID:24780083

Production of cellulosic ethanol from sugarcane bagasse by steam explosion: Effect of extractives content, acid catalysis and different fermentation technologies.

PubMed

Neves, P V; Pitarelo, A P; Ramos, L P

2016-05-01

The production of cellulosic ethanol was carried out using samples of native (NCB) and ethanol-extracted (EECB) sugarcane bagasse. Autohydrolysis (AH) exhibited the best glucose recovery from both samples, compared to the use of both H3PO4 and H2SO4 catalysis at the same pretreatment time and temperature. All water-insoluble steam-exploded materials (SEB-WI) resulted in high glucose yields by enzymatic hydrolysis. SHF (separate hydrolysis and fermentation) gave ethanol yields higher than those obtained by SSF (simultaneous hydrolysis and fermentation) and pSSF (pre-hydrolysis followed by SSF). For instance, AH gave 25, 18 and 16 g L(-1) of ethanol by SHF, SSF and pSSF, respectively. However, when the total processing time was taken into account, pSSF provided the best overall ethanol volumetric productivity of 0.58 g L(-1) h(-1). Also, the removal of ethanol-extractable materials from cane bagasse had no influence on the cellulosic ethanol production of SEB-WI, regardless of the fermentation strategy used for conversion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heterogeneous Catalysis.

ERIC Educational Resources Information Center

Vannice, M. A.

1979-01-01

Described is a graduate course in catalysis offered at Penn State University. A detailed course outline with 30 lecture topics is presented. A list of 42 references on catalysis used in place of a textbook is provided. (BT)

Asymmetric Fluorination of α-Branched Cyclohexanones Enabled by a Combination of Chiral Anion Phase-Transfer Catalysis and Enamine Catalysis using Protected Amino Acids

PubMed Central

2015-01-01

We report a study involving the successful merger of two separate chiral catalytic cycles: a chiral anion phase-transfer catalysis cycle to activate Selectfluor and an enamine activation cycle, using a protected amino acid as organocatalyst. We have demonstrated the viability of this approach with the direct asymmetric fluorination of α-substituted cyclohexanones to generate quaternary fluorine-containing stereocenters. With these two chiral catalytic cycles operating together in a matched sense, high enantioselectivites can be achieved, and we envisage that this dual catalysis method has the potential to be more broadly applicable, given the breadth of enamine catalysis. It also represents a rare example of chiral enamine catalysis operating successfully on α-branched ketones, substrates commonly inert to this activation mode. PMID:24684209

Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

PubMed

Pachov, Dimitar V; van den Bedem, Henry

2015-07-01

Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA) report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS) permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs) is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key structural elements of Gαs.

Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs

PubMed Central

Pachov, Dimitar V.; van den Bedem, Henry

2015-01-01

Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA) report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS) permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs) is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key structural elements of Gαs. PMID:26218073

Laboratory evolution of protein conformational dynamics.

PubMed

Campbell, Eleanor C; Correy, Galen J; Mabbitt, Peter D; Buckle, Ashley M; Tokuriki, Nobuhiko; Jackson, Colin J

2017-11-08

This review focuses on recent work that has begun to establish specific functional roles for protein conformational dynamics, specifically how the conformational landscapes that proteins can sample can evolve under laboratory based evolutionary selection. We discuss recent technical advances in computational and biophysical chemistry, which have provided us with new ways to dissect evolutionary processes. Finally, we offer some perspectives on the emerging view of conformational dynamics and evolution, and the challenges that we face in rationally engineering conformational dynamics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Accelerating the Conformational Sampling of Intrinsically Disordered Proteins.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2014-11-11

Intrinsically disordered proteins (IDPs) are a class of proteins lacking a well-defined secondary structure. Instead, they are able to attain multiple conformations, bind to multiple targets, and respond to changes in their surroundings. Functionally, IDPs have been associated with molecular recognition, cell regulation, and signal transduction. The dynamic conformational ensemble of IDPs is highly environmental and binding partner dependent, rendering the characterization of IDPs extremely challenging. Here, we compare the sampling efficiencies of conventional molecular dynamics (MD), well-tempered metadynamics (WT-META), and bias-exchange metadynamics (BE-META). The total simulation time was over 10 μs, and a 20-mer peptide derived from the Neh2 domain of the Nuclear factor erythroid 2-related factor 2 (Nrf2) protein was simulated. BE-META, with a neutral replica and seven biased replicas employing a set of seven relevant collective variables (CVs), provided the most reliable and efficient sampling. Finally, we propose a free-energy reconstruction method based on the probability distribution of the secondary structure contents. This postprocessing analysis confirms the presence of not only the β-hairpin conformation of the free Neh2 peptide but also its rare bound-state-like conformation, both of that have been experimentally observed. In addition, our simulations also predict other possible conformations to be verified with future experiments.

Cosmic strings and baryon decay catalysis

NASA Technical Reports Server (NTRS)

Gregory, Ruth; Perkins, W. B.; Davis, A.-C.; Brandenberger, R. H.

1989-01-01

Cosmic strings, like monopoles, can catalyze proton decay. For integer charged fermions, the cross section for catalysis is not amplified, unlike in the case of monopoles. The catalysis processes are reviewed both in the free quark and skyrmion pictures and the implications for baryogenesis are discussed. A computation of the cross section for monopole catalyzed skyrmion decay is presented using classical physics. Also discussed are some effects which can screen catalysis processes.

NMR Studies of Dynamic Biomolecular Conformational Ensembles

PubMed Central

Torchia, Dennis A.

2015-01-01

Multidimensional heteronuclear NMR approaches can provide nearly complete sequential signal assignments of isotopically enriched biomolecules. The availability of assignments together with measurements of spin relaxation rates, residual spin interactions, J-couplings and chemical shifts provides information at atomic resolution about internal dynamics on timescales ranging from ps to ms, both in solution and in the solid state. However, due to the complexity of biomolecules, it is not possible to extract a unique atomic-resolution description of biomolecular motions even from extensive NMR data when many conformations are sampled on multiple timescales. For this reason, powerful computational approaches are increasingly applied to large NMR data sets to elucidate conformational ensembles sampled by biomolecules. In the past decade, considerable attention has been directed at an important class of biomolecules that function by binding to a wide variety of target molecules. Questions of current interest are: “Does the free biomolecule sample a conformational ensemble that encompasses the conformations found when it binds to various targets; and if so, on what time scale is the ensemble sampled?” This article reviews recent efforts to answer these questions, with a focus on comparing ensembles obtained for the same biomolecules by different investigators. A detailed comparison of results obtained is provided for three biomolecules: ubiquitin, calmodulin and the HIV-1 trans-activation response RNA. PMID:25669739

Support for U.S. Participants at the 15th International Congress on Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wachs, Israel E.

2013-08-05

The grant was used to partially assist the travel expenses of U.S. academic scientists to attend the 15th International Congress on Catalysis. The conference was held in Munich, Germany from July 1-6, 2012. The importance of the International Congress was to bring together the international community of faculty members who participate in catalysis research, and to share information that would lead to further developments and directions in the field of study. Graduate students and Post Docs were invited to apply for travel assistance based on criteria established by the North American Catalysis Society (NACS) and the local Catalysis Clubs.

Catalysis of Photochemical Reactions.

ERIC Educational Resources Information Center

Albini, A.

1986-01-01

Offers a classification system of catalytic effects in photochemical reactions, contrasting characteristic properties of photochemical and thermal reactions. Discusses catalysis and sensitization, examples of catalyzed reactions of excepted states, complexing ground state substrates, and catalysis of primary photoproducts. (JM)

Enhanced and effective conformational sampling of protein molecular systems for their free energy landscapes.

PubMed

Higo, Junichi; Ikebe, Jinzen; Kamiya, Narutoshi; Nakamura, Haruki

2012-03-01

Protein folding and protein-ligand docking have long persisted as important subjects in biophysics. Using multicanonical molecular dynamics (McMD) simulations with realistic expressions, i.e., all-atom protein models and an explicit solvent, free-energy landscapes have been computed for several systems, such as the folding of peptides/proteins composed of a few amino acids up to nearly 60 amino-acid residues, protein-ligand interactions, and coupled folding and binding of intrinsically disordered proteins. Recent progress in conformational sampling and its applications to biophysical systems are reviewed in this report, including descriptions of several outstanding studies. In addition, an algorithm and detailed procedures used for multicanonical sampling are presented along with the methodology of adaptive umbrella sampling. Both methods control the simulation so that low-probability regions along a reaction coordinate are sampled frequently. The reaction coordinate is the potential energy for multicanonical sampling and is a structural identifier for adaptive umbrella sampling. One might imagine that this probability control invariably enhances conformational transitions among distinct stable states, but this study examines the enhanced conformational sampling of a simple system and shows that reasonably well-controlled sampling slows the transitions. This slowing is induced by a rapid change of entropy along the reaction coordinate. We then provide a recipe to speed up the sampling by loosening the rapid change of entropy. Finally, we report all-atom McMD simulation results of various biophysical systems in an explicit solvent.

High-efficiency and mechano-/photo- bi-catalysis of piezoelectric-ZnO@ photoelectric-TiO2 core-shell nanofibers for dye decomposition.

PubMed

You, Huilin; Wu, Zheng; Jia, Yanmin; Xu, Xiaoli; Xia, Yuntao; Han, Zichen; Wang, Yu

2017-09-01

A mechano-/photo- bi-catalyst of piezoelectric-ZnO@photoelectric-TiO 2 core-shell nanofibers was hydrothermally synthesized for Methyl Orange (10 mg L -1 ) decomposition. The mechano-/photo- bi-catalysis in ZnO@TiO 2 is superior to mechano- or photo-catalysis in decomposing Methyl Orange, which is mainly attributed to the synergy effect of the piezoelectric-ZnO core's mechano-catalysis and the thin photoelectric TiO 2 shell's photo-catalysis. The heterostructure of the piezoelectric-ZnO@photoelectric-TiO 2 core-shell interface, being helpful to reduce electron-hole pair recombination and to separate the piezoelectrically-/photoelectric ally- induced electrons and holes, may also make a great contribution to the enhanced catalysis performance. The mechano-/photo-bi-catalysis in ZnO@TiO 2 core-shell nanofibers possesses the advantages of high efficiency, non-toxicity and tractability and is potential in utilizing mechanical/solar energy to deal with dye wastewater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modulation of individual steps in group I intron catalysis by a peripheral metal ion.

PubMed

Forconi, Marcello; Piccirilli, Joseph A; Herschlag, Daniel

2007-10-01

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

Allosteric modulation of Ras positions Q61 for a direct role in catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; Holzapfel, Genevieve; Fetics, Susan

2010-11-03

Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the {gamma}-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Rasmore » with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the {gamma}-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca{sup 2+} and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.« less

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals

DOE PAGES

Peck, Ariana; Poitevin, Frederic; Lane, Thomas Joseph

2018-02-21

Conformational changes drive protein function, including catalysis, allostery, and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signalmore » reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.« less

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'–3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed endsmore » at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.« less

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I.

PubMed

Shi, Yuqian; Hellinga, Homme W; Beese, Lorena S

2017-06-06

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.

Specificity of Processing α-glucosidase I is guided by the substrate conformation: crystallographic and in silico studies.

PubMed

Barker, Megan K; Rose, David R

2013-05-10

The enzyme “GluI” is key to the synthesis of critical glycoproteins in the cell. We have determined the structure of GluI, and modeled binding with its unique sugar substrate. The specificity of this interaction derives from a unique conformation of the substrate. Understanding the mechanism of the enzyme is of basic importance and relevant to potential development of antiviral inhibitors. Processing α-glucosidase I (GluI) is a key member of the eukaryotic N-glycosylation processing pathway, selectively catalyzing the first glycoprotein trimming step in the endoplasmic reticulum. Inhibition of GluI activity impacts the infectivity of enveloped viruses; however, despite interest in this protein from a structural, enzymatic, and therapeutic standpoint, little is known about its structure and enzymatic mechanism in catalysis of the unique glycan substrate Glc3Man9GlcNAc2. The first structural model of eukaryotic GluI is here presented at 2-Å resolution. Two catalytic residues are proposed, mutations of which result in catalytically inactive, properly folded protein. Using Autodocking methods with the known substrate and inhibitors as ligands, including a novel inhibitor characterized in this work, the active site of GluI was mapped. From these results, a model of substrate binding has been formulated, which is most likely conserved in mammalian GluI.

Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

PubMed

Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

2017-05-01

Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

Intermolecular correlations are necessary to explain diffuse scattering from protein crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peck, Ariana; Poitevin, Frederic; Lane, Thomas Joseph

Conformational changes drive protein function, including catalysis, allostery, and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signalmore » reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.« less

The Physics and Physical Chemistry of Molecular Machines.

PubMed

Astumian, R Dean; Mukherjee, Shayantani; Warshel, Arieh

2016-06-17

The concept of a "power stroke"-a free-energy releasing conformational change-appears in almost every textbook that deals with the molecular details of muscle, the flagellar rotor, and many other biomolecular machines. Here, it is shown by using the constraints of microscopic reversibility that the power stroke model is incorrect as an explanation of how chemical energy is used by a molecular machine to do mechanical work. Instead, chemically driven molecular machines operating under thermodynamic constraints imposed by the reactant and product concentrations in the bulk function as information ratchets in which the directionality and stopping torque or stopping force are controlled entirely by the gating of the chemical reaction that provides the fuel for the machine. The gating of the chemical free energy occurs through chemical state dependent conformational changes of the molecular machine that, in turn, are capable of generating directional mechanical motions. In strong contrast to this general conclusion for molecular machines driven by catalysis of a chemical reaction, a power stroke may be (and often is) an essential component for a molecular machine driven by external modulation of pH or redox potential or by light. This difference between optical and chemical driving properties arises from the fundamental symmetry difference between the physics of optical processes, governed by the Bose-Einstein relations, and the constraints of microscopic reversibility for thermally activated processes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

PubMed

Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Computational Design of Clusters for Catalysis

NASA Astrophysics Data System (ADS)

Jimenez-Izal, Elisa; Alexandrova, Anastassia N.

2018-04-01

When small clusters are studied in chemical physics or physical chemistry, one perhaps thinks of the fundamental aspects of cluster electronic structure, or precision spectroscopy in ultracold molecular beams. However, small clusters are also of interest in catalysis, where the cold ground state or an isolated cluster may not even be the right starting point. Instead, the big question is: What happens to cluster-based catalysts under real conditions of catalysis, such as high temperature and coverage with reagents? Myriads of metastable cluster states become accessible, the entire system is dynamic, and catalysis may be driven by rare sites present only under those conditions. Activity, selectivity, and stability are highly dependent on size, composition, shape, support, and environment. To probe and master cluster catalysis, sophisticated tools are being developed for precision synthesis, operando measurements, and multiscale modeling. This review intends to tell the messy story of clusters in catalysis.

NMSim web server: integrated approach for normal mode-based geometric simulations of biologically relevant conformational transitions in proteins.

PubMed

Krüger, Dennis M; Ahmed, Aqeel; Gohlke, Holger

2012-07-01

The NMSim web server implements a three-step approach for multiscale modeling of protein conformational changes. First, the protein structure is coarse-grained using the FIRST software. Second, a rigid cluster normal-mode analysis provides low-frequency normal modes. Third, these modes are used to extend the recently introduced idea of constrained geometric simulations by biasing backbone motions of the protein, whereas side chain motions are biased toward favorable rotamer states (NMSim). The generated structures are iteratively corrected regarding steric clashes and stereochemical constraint violations. The approach allows performing three simulation types: unbiased exploration of conformational space; pathway generation by a targeted simulation; and radius of gyration-guided simulation. On a data set of proteins with experimentally observed conformational changes, the NMSim approach has been shown to be a computationally efficient alternative to molecular dynamics simulations for conformational sampling of proteins. The generated conformations and pathways of conformational transitions can serve as input to docking approaches or more sophisticated sampling techniques. The web server output is a trajectory of generated conformations, Jmol representations of the coarse-graining and a subset of the trajectory and data plots of structural analyses. The NMSim webserver, accessible at http://www.nmsim.de, is free and open to all users with no login requirement.

A Free-Energy Approach for All-Atom Protein Simulation

PubMed Central

Verma, Abhinav; Wenzel, Wolfgang

2009-01-01

All-atom free-energy methods offer a promising alternative to kinetic molecular mechanics simulations of protein folding and association. Here we report an accurate, transferable all-atom biophysical force field (PFF02) that stabilizes the native conformation of a wide range of proteins as the global optimum of the free-energy landscape. For 32 proteins of the ROSETTA decoy set and six proteins that we have previously folded with PFF01, we find near-native conformations with an average backbone RMSD of 2.14 Å to the native conformation and an average Z-score of −3.46 to the corresponding decoy set. We used nonequilibrium sampling techniques starting from completely extended conformations to exhaustively sample the energy surface of three nonhomologous hairpin-peptides, a three-stranded β-sheet, the all-helical 40 amino-acid HIV accessory protein, and a zinc-finger ββα motif, and find near-native conformations for the minimal energy for each protein. Using a massively parallel evolutionary algorithm, we also obtain a near-native low-energy conformation for the 54 amino-acid engrailed homeodomain. Our force field thus stabilized near-native conformations for a total of 20 proteins of all structure classes with an average RMSD of only 3.06 Å to their respective experimental conformations. PMID:19413955

A free-energy approach for all-atom protein simulation.

PubMed

Verma, Abhinav; Wenzel, Wolfgang

2009-05-06

All-atom free-energy methods offer a promising alternative to kinetic molecular mechanics simulations of protein folding and association. Here we report an accurate, transferable all-atom biophysical force field (PFF02) that stabilizes the native conformation of a wide range of proteins as the global optimum of the free-energy landscape. For 32 proteins of the ROSETTA decoy set and six proteins that we have previously folded with PFF01, we find near-native conformations with an average backbone RMSD of 2.14 A to the native conformation and an average Z-score of -3.46 to the corresponding decoy set. We used nonequilibrium sampling techniques starting from completely extended conformations to exhaustively sample the energy surface of three nonhomologous hairpin-peptides, a three-stranded beta-sheet, the all-helical 40 amino-acid HIV accessory protein, and a zinc-finger beta beta alpha motif, and find near-native conformations for the minimal energy for each protein. Using a massively parallel evolutionary algorithm, we also obtain a near-native low-energy conformation for the 54 amino-acid engrailed homeodomain. Our force field thus stabilized near-native conformations for a total of 20 proteins of all structure classes with an average RMSD of only 3.06 A to their respective experimental conformations.

Multiple Replica Repulsion Technique for Efficient Conformational Sampling of Biological Systems

PubMed Central

Malevanets, Anatoly; Wodak, Shoshana J.

2011-01-01

Here, we propose a technique for sampling complex molecular systems with many degrees of freedom. The technique, termed “multiple replica repulsion” (MRR), does not suffer from poor scaling with the number of degrees of freedom associated with common replica exchange procedures and does not require sampling at high temperatures. The algorithm involves creation of multiple copies (replicas) of the system, which interact with one another through a repulsive potential that can be applied to the system as a whole or to portions of it. The proposed scheme prevents oversampling of the most populated states and provides accurate descriptions of conformational perturbations typically associated with sampling ground-state energy wells. The performance of MRR is illustrated for three systems of increasing complexity. A two-dimensional toy potential surface is used to probe the sampling efficiency as a function of key parameters of the procedure. MRR simulations of the Met-enkephalin pentapeptide, and the 76-residue protein ubiquitin, performed in presence of explicit water molecules and totaling 32 ns each, investigate the ability of MRR to characterize the conformational landscape of the peptide, and the protein native basin, respectively. Results obtained for the enkephalin peptide reflect more closely the extensive conformational flexibility of this peptide than previously reported simulations. Those obtained for ubiquitin show that conformational ensembles sampled by MRR largely encompass structural fluctuations relevant to biological recognition, which occur on the microsecond timescale, or are observed in crystal structures of ubiquitin complexes with other proteins. MRR thus emerges as a very promising simple and versatile technique for modeling the structural plasticity of complex biological systems. PMID:21843487

Predictive Sampling of Rare Conformational Events in Aqueous Solution: Designing a Generalized Orthogonal Space Tempering Method.

PubMed

Lu, Chao; Li, Xubin; Wu, Dongsheng; Zheng, Lianqing; Yang, Wei

2016-01-12

In aqueous solution, solute conformational transitions are governed by intimate interplays of the fluctuations of solute-solute, solute-water, and water-water interactions. To promote molecular fluctuations to enhance sampling of essential conformational changes, a common strategy is to construct an expanded Hamiltonian through a series of Hamiltonian perturbations and thereby broaden the distribution of certain interactions of focus. Due to a lack of active sampling of configuration response to Hamiltonian transitions, it is challenging for common expanded Hamiltonian methods to robustly explore solvent mediated rare conformational events. The orthogonal space sampling (OSS) scheme, as exemplified by the orthogonal space random walk and orthogonal space tempering methods, provides a general framework for synchronous acceleration of slow configuration responses. To more effectively sample conformational transitions in aqueous solution, in this work, we devised a generalized orthogonal space tempering (gOST) algorithm. Specifically, in the Hamiltonian perturbation part, a solvent-accessible-surface-area-dependent term is introduced to implicitly perturb near-solute water-water fluctuations; more importantly in the orthogonal space response part, the generalized force order parameter is generalized as a two-dimension order parameter set, in which essential solute-solvent and solute-solute components are separately treated. The gOST algorithm is evaluated through a molecular dynamics simulation study on the explicitly solvated deca-alanine (Ala10) peptide. On the basis of a fully automated sampling protocol, the gOST simulation enabled repetitive folding and unfolding of the solvated peptide within a single continuous trajectory and allowed for detailed constructions of Ala10 folding/unfolding free energy surfaces. The gOST result reveals that solvent cooperative fluctuations play a pivotal role in Ala10 folding/unfolding transitions. In addition, our assessment analysis suggests that because essential conformational events are mainly driven by the compensating fluctuations of essential solute-solvent and solute-solute interactions, commonly employed "predictive" sampling methods are unlikely to be effective on this seemingly "simple" system. The gOST development presented in this paper illustrates how to employ the OSS scheme for physics-based sampling method designs.

Kinetics and Catalysis Demonstrations.

ERIC Educational Resources Information Center

Falconer, John L.; Britten, Jerald A.

1984-01-01

Eleven videotaped kinetics and catalysis demonstrations are described. Demonstrations include the clock reaction, oscillating reaction, hydrogen oxidation in air, hydrogen-oxygen explosion, acid-base properties of solids, high- and low-temperature zeolite reactivity, copper catalysis of ammonia oxidation and sodium peroxide decomposition, ammonia…

Probabilistic sampling of protein conformations: new hope for brute force?

PubMed

Feldman, Howard J; Hogue, Christopher W V

2002-01-01

Protein structure prediction from sequence alone by "brute force" random methods is a computationally expensive problem. Estimates have suggested that it could take all the computers in the world longer than the age of the universe to compute the structure of a single 200-residue protein. Here we investigate the use of a faster version of our FOLDTRAJ probabilistic all-atom protein-structure-sampling algorithm. We have improved the method so that it is now over twenty times faster than originally reported, and capable of rapidly sampling conformational space without lattices. It uses geometrical constraints and a Leonard-Jones type potential for self-avoidance. We have also implemented a novel method to add secondary structure-prediction information to make protein-like amounts of secondary structure in sampled structures. In a set of 100,000 probabilistic conformers of 1VII, 1ENH, and 1PMC generated, the structures with smallest Calpha RMSD from native are 3.95, 5.12, and 5.95A, respectively. Expanding this test to a set of 17 distinct protein folds, we find that all-helical structures are "hit" by brute force more frequently than beta or mixed structures. For small helical proteins or very small non-helical ones, this approach should have a "hit" close enough to detect with a good scoring function in a pool of several million conformers. By fitting the distribution of RMSDs from the native state of each of the 17 sets of conformers to the extreme value distribution, we are able to estimate the size of conformational space for each. With a 0.5A RMSD cutoff, the number of conformers is roughly 2N where N is the number of residues in the protein. This is smaller than previous estimates, indicating an average of only two possible conformations per residue when sterics are accounted for. Our method reduces the effective number of conformations available at each residue by probabilistic bias, without requiring any particular discretization of residue conformational space, and is the fastest method of its kind. With computer speeds doubling every 18 months and parallel and distributed computing becoming more practical, the brute force approach to protein structure prediction may yet have some hope in the near future. Copyright 2001 Wiley-Liss, Inc.

Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.

PubMed

Goryanova, Bogdana; Goldman, Lawrence M; Ming, Shonoi; Amyes, Tina L; Gerlt, John A; Richard, John P

2015-07-28

The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Virtual special issue on catalysis at the U.S. Department of Energy's National Laboratories

DOE PAGES

Pruski, Marek; Sadow, Aaron D.; Slowing, Igor I.; ...

2016-04-21

Here the catalysis research at the U.S. Department of Energy's (DOE's) National Laboratories covers a wide range of research topics in heterogeneous catalysis, homogeneous/molecular catalysis, biocatalysis, electrocatalysis, and surface science. Since much of the work at National Laboratories is funded by DOE, the research is largely focused on addressing DOE's mission to ensure America's security and prosperity by addressing its energy, environmental, and nuclear challenges through transformative science and technology solutions.

Heterogeneous Molecular Catalysis of Electrochemical Reactions: Volcano Plots and Catalytic Tafel Plots.

PubMed

Costentin, Cyrille; Savéant, Jean-Michel

2017-06-14

We analyze here, in the framework of heterogeneous molecular catalysis, the reasons for the occurrence or nonoccurrence of volcanoes upon plotting the kinetics of the catalytic reaction versus the stabilization free energy of the primary intermediate of the catalytic process. As in the case of homogeneous molecular catalysis or catalysis by surface-active metallic sites, a strong motivation of such studies relates to modern energy challenges, particularly those involving small molecules, such as water, hydrogen, oxygen, proton, and carbon dioxide. This motivation is particularly pertinent for what concerns heterogeneous molecular catalysis, since it is commonly preferred to homogeneous molecular catalysis by the same molecules if only for chemical separation purposes and electrolytic cell architecture. As with the two other catalysis modes, the main drawback of the volcano plot approach is the basic assumption that the kinetic responses depend on a single descriptor, viz., the stabilization free energy of the primary intermediate. More comprehensive approaches, investigating the responses to the maximal number of experimental factors, and conveniently expressed as catalytic Tafel plots, should clearly be preferred. This is more so in the case of heterogeneous molecular catalysis in that additional transport factors in the supporting film may additionally affect the current-potential responses. This is attested by the noteworthy presence of maxima in catalytic Tafel plots as well as their dependence upon the cyclic voltammetric scan rate.

Free energy landscapes of peptides by enhanced conformational sampling.

PubMed

Nakajima, N; Higo, J; Kidera, A; Nakamura, H

2000-02-11

The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors. Copyright 2000 Academic Press.

Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study.

PubMed

Zhao, Yuan; She, Nai; Zhang, Xin; Wang, Chaojie; Mo, Yirong

2017-08-01

Yeast cytosine deaminase (yCD) is critical in gene-directed enzyme prodrug therapy as it catalyzes the hydrolytic deamination of cytosine. The product (uracil) release process is considered as rate-limiting in the whole enzymatic catalysis and includes the cleavage of the uracil-metal bond and the delivery of free uracil out of the reactive site. Herein extensive combined random acceleration molecular dynamics (RAMD) and molecular dynamics (MD) simulations coupled with the umbrella sampling technique have been performed to study the product transport mechanism. Five channels have been identified, and the thermodynamic and dynamic characterizations for the two most favorable channels have been determined and analyzed. The free energy barrier for the most beneficial pathway is about 13kcal/mol and mainly results from the cleavage of hydrogen bonds between the ligand uracil and surrounding residues Asn51, Glu64, and Asp155. The conjugated rings of Phe114 and Trp152 play gating and guiding roles in the product delivery via π⋯π van der Waals interactions with the product. Finally, the full cycle of the enzymatic catalysis has been determined, making the whole process computationally more precise. Copyright © 2017 Elsevier B.V. All rights reserved.

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.

PubMed

Sullivan, Sarah M; Holyoak, Todd

2007-09-04

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3.

PubMed

Nowroozi, Amin; Shahlaei, Mohsen

2017-02-01

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.

De novo protein structure prediction by dynamic fragment assembly and conformational space annealing.

PubMed

Lee, Juyong; Lee, Jinhyuk; Sasaki, Takeshi N; Sasai, Masaki; Seok, Chaok; Lee, Jooyoung

2011-08-01

Ab initio protein structure prediction is a challenging problem that requires both an accurate energetic representation of a protein structure and an efficient conformational sampling method for successful protein modeling. In this article, we present an ab initio structure prediction method which combines a recently suggested novel way of fragment assembly, dynamic fragment assembly (DFA) and conformational space annealing (CSA) algorithm. In DFA, model structures are scored by continuous functions constructed based on short- and long-range structural restraint information from a fragment library. Here, DFA is represented by the full-atom model by CHARMM with the addition of the empirical potential of DFIRE. The relative contributions between various energy terms are optimized using linear programming. The conformational sampling was carried out with CSA algorithm, which can find low energy conformations more efficiently than simulated annealing used in the existing DFA study. The newly introduced DFA energy function and CSA sampling algorithm are implemented into CHARMM. Test results on 30 small single-domain proteins and 13 template-free modeling targets of the 8th Critical Assessment of protein Structure Prediction show that the current method provides comparable and complementary prediction results to existing top methods. Copyright © 2011 Wiley-Liss, Inc.

Visualization of SV2A conformations in situ by the use of Protein Tomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lynch, Berkley A.; Matagne, Alain; Braennstroem, Annika

2008-10-31

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography{sup TM}. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicatesmore » that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.« less

Chemical Synthesis of Complex Molecules Using Nanoparticle Catalysis

PubMed Central

Cong, Huan; Porco, John A.

2011-01-01

Nanoparticle catalysis has emerged as an active topic in organic synthesis. Of particular interest is the development of enabling methodologies to efficiently assemble complex molecules using nanoparticle catalysis. This Viewpoint highlights recent developments and discusses future perspectives in this emerging field. PMID:22347681

Daniel Dupuis | NREL

Science.gov Websites

Dupuis Dan Dupuis Advanced Catalysis Engineer Daniel.Dupuis@nrel.gov | 303-384-7685 Orcid ID http ://orcid.org/0000-0002-1142-9713 Research Interests Advanced fuel synthesis and catalysis Catalyst synthesis Community College, 2012 Professional Experience Advanced Catalysis Engineer, National Renewable Energy

Catalysis and prebiotic RNA synthesis

NASA Technical Reports Server (NTRS)

Ferris, James P.

1993-01-01

The essential role of catalysis for the origins of life is discussed. The status of the prebiotic synthesis of 2',5'- and 3'5'-linked oligomers of RNA is reviewed. Examples of the role of metal ion and mineral catalysis in RNA oligomer formation are discussed.

Sequential rhodium/palladium catalysis: enantioselective formation of dihydroquinolinones in the presence of achiral and chiral ligands.

PubMed

Zhang, Lei; Qureshi, Zafar; Sonaglia, Lorenzo; Lautens, Mark

2014-12-08

Compatible combinations of achiral and chiral ligands can be used in rhodium/palladium catalysis to achieve highly enantioselective domino reactions. The difference in rates of catalysis and minimal effects of ligand interference confer control in the domino sequence. The "all-in-one" 1,4-conjugate arylation and C-N cross-coupling through sequential Rh/Pd catalysis provides access to enantioenriched dihydroquinolinone building blocks. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced Conformational Sampling Using Replica Exchange with Collective-Variable Tempering.

PubMed

Gil-Ley, Alejandro; Bussi, Giovanni

2015-03-10

The computational study of conformational transitions in RNA and proteins with atomistic molecular dynamics often requires suitable enhanced sampling techniques. We here introduce a novel method where concurrent metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strengths of the bias potential exploiting the tunability of well-tempered metadynamics. Using this method, free-energy barriers of individual collective variables are significantly reduced compared with simple force-field scaling. The introduced methodology is flexible and allows adaptive bias potentials to be self-consistently constructed for a large number of simple collective variables, such as distances and dihedral angles. The method is tested on alanine dipeptide and applied to the difficult problem of conformational sampling in a tetranucleotide.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

DOE PAGES

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas; ...

2017-08-23

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

Procrustean rotation in concert with principal component analysis of molecular dynamics trajectories: Quantifying global and local differences between conformational samples.

PubMed

Oblinsky, Daniel G; Vanschouwen, Bryan M B; Gordon, Heather L; Rothstein, Stuart M

2009-12-14

Given the principal component analysis (PCA) of a molecular dynamics (MD) conformational trajectory for a model protein, we perform orthogonal Procrustean rotation to "best fit" the PCA squared-loading matrix to that of a target matrix computed for a related but different molecular system. The sum of squared deviations of the elements of the rotated matrix from those of the target, known as the error of fit (EOF), provides a quantitative measure of the dissimilarity between the two conformational samples. To estimate precision of the EOF, we perform bootstrap resampling of the molecular conformations within the trajectories, generating a distribution of EOF values for the system and target. The average EOF per variable is determined and visualized to ascertain where, locally, system and target sample properties differ. We illustrate this approach by analyzing MD trajectories for the wild-type and four selected mutants of the beta1 domain of protein G.

Procrustean rotation in concert with principal component analysis of molecular dynamics trajectories: Quantifying global and local differences between conformational samples

NASA Astrophysics Data System (ADS)

Oblinsky, Daniel G.; VanSchouwen, Bryan M. B.; Gordon, Heather L.; Rothstein, Stuart M.

2009-12-01

Given the principal component analysis (PCA) of a molecular dynamics (MD) conformational trajectory for a model protein, we perform orthogonal Procrustean rotation to "best fit" the PCA squared-loading matrix to that of a target matrix computed for a related but different molecular system. The sum of squared deviations of the elements of the rotated matrix from those of the target, known as the error of fit (EOF), provides a quantitative measure of the dissimilarity between the two conformational samples. To estimate precision of the EOF, we perform bootstrap resampling of the molecular conformations within the trajectories, generating a distribution of EOF values for the system and target. The average EOF per variable is determined and visualized to ascertain where, locally, system and target sample properties differ. We illustrate this approach by analyzing MD trajectories for the wild-type and four selected mutants of the β1 domain of protein G.

Enhanced conformational sampling technique provides an energy landscape view of large-scale protein conformational transitions.

PubMed

Shao, Qiang

2016-10-26

Large-scale conformational changes in proteins are important for their functions. Tracking the conformational change in real time at the level of a single protein molecule, however, remains a great challenge. In this article, we present a novel in silico approach with the combination of normal mode analysis and integrated-tempering-sampling molecular simulation (NMA-ITS) to give quantitative data for exploring the conformational transition pathway in multi-dimensional energy landscapes starting only from the knowledge of the two endpoint structures of the protein. The open-to-closed transitions of three proteins, including nCaM, AdK, and HIV-1 PR, were investigated using NMA-ITS simulations. The three proteins have varied structural flexibilities and domain communications in their respective conformational changes. The transition state structure in the conformational change of nCaM and the associated free-energy barrier are in agreement with those measured in a standard explicit-solvent REMD simulation. The experimentally measured transition intermediate structures of the intrinsically flexible AdK are captured by the conformational transition pathway measured here. The dominant transition pathways between the closed and fully open states of HIV-1 PR are very similar to those observed in recent REMD simulations. Finally, the evaluated relaxation times of the conformational transitions of three proteins are roughly at the same level as reported experimental data. Therefore, the NMA-ITS method is applicable for a variety of cases, providing both qualitative and quantitative insights into the conformational changes associated with the real functions of proteins.

Enantio-Relay Catalysis Constructs Chiral Biaryl Alcohols over Cascade Suzuki Cross-Coupling-Asymmetric Transfer Hydrogenation

NASA Astrophysics Data System (ADS)

Zhang, Dacheng; Gao, Xiaoshuang; Cheng, Tanyu; Liu, Guohua

2014-05-01

The construction of chiral biaryl alcohols using enantio-relay catalysis is a particularly attractive synthetic method in organic synthesis. However, overcoming the intrinsic incompatibility of distinct organometallic complexes and the reaction conditions used are significant challenges in asymmetric catalysis. To overcome these barriers, we have taken advantage of an enantio-relay catalysis strategy and a combined dual-immobilization approach. We report the use of an imidazolium-based organopalladium-functionalized organic-inorganic hybrid silica and ethylene-coated chiral organoruthenium-functionalized magnetic nanoparticles to catalyze a cascade Suzuki cross-coupling-asymmetric transfer hydrogenation reaction to prepare chiral biaryl alcohols in a two-step, one-pot process. As expected, the site-isolated active species, salient imidazolium phase-transfer character and high ethylene-coated hydrophobicity can synergistically boost the catalytic performance. Furthermore, enantio-relay catalysis has the potential to efficiently prepare a variety of chiral biaryl alcohols. Our synthetic strategy is a general method that shows the potential of developing enantio-relay catalysis towards environmentally benign and sustainable organic synthesis.

Unique Aspects of the Structure and Dynamics of Elementary Iβ Cellulose Microfibrils Revealed by Computational Simulations1[OPEN

PubMed Central

Oehme, Daniel P.; Downton, Matthew T.; Doblin, Monika S.; Wagner, John; Gidley, Michael J.; Bacic, Antony

2015-01-01

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Iβ cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains. PMID:25786828

Exploring transition pathway and free-energy profile of large-scale protein conformational change by combining normal mode analysis and umbrella sampling molecular dynamics.

PubMed

Wang, Jinan; Shao, Qiang; Xu, Zhijian; Liu, Yingtao; Yang, Zhuo; Cossins, Benjamin P; Jiang, Hualiang; Chen, Kaixian; Shi, Jiye; Zhu, Weiliang

2014-01-09

Large-scale conformational changes of proteins are usually associated with the binding of ligands. Because the conformational changes are often related to the biological functions of proteins, understanding the molecular mechanisms of these motions and the effects of ligand binding becomes very necessary. In the present study, we use the combination of normal-mode analysis and umbrella sampling molecular dynamics simulation to delineate the atomically detailed conformational transition pathways and the associated free-energy landscapes for three well-known protein systems, viz., adenylate kinase (AdK), calmodulin (CaM), and p38α kinase in the absence and presence of respective ligands. For each protein under study, the transient conformations along the conformational transition pathway and thermodynamic observables are in agreement with experimentally and computationally determined ones. The calculated free-energy profiles reveal that AdK and CaM are intrinsically flexible in structures without obvious energy barrier, and their ligand binding shifts the equilibrium from the ligand-free to ligand-bound conformation (population shift mechanism). In contrast, the ligand binding to p38α leads to a large change in free-energy barrier (ΔΔG ≈ 7 kcal/mol), promoting the transition from DFG-in to DFG-out conformation (induced fit mechanism). Moreover, the effect of the protonation of D168 on the conformational change of p38α is also studied, which reduces the free-energy difference between the two functional states of p38α and thus further facilitates the conformational interconversion. Therefore, the present study suggests that the detailed mechanism of ligand binding and the associated conformational transition is not uniform for all kinds of proteins but correlated to their respective biological functions.

Toward a general mixed quantum/classical method for the calculation of the vibronic ECD of a flexible dye molecule with different stable conformers: Revisiting the case of 2,2,2-trifluoro-anthrylethanol.

PubMed

Cerezo, Javier; Aranda, Daniel; Avila Ferrer, Francisco J; Prampolini, Giacomo; Mazzeo, Giuseppe; Longhi, Giovanna; Abbate, Sergio; Santoro, Fabrizio

2018-06-01

We extend a recently proposed mixed quantum/classical method for computing the vibronic electronic circular dichroism (ECD) spectrum of molecules with different conformers, to cases where more than one hindered rotation is present. The method generalizes the standard procedure, based on the simple Boltzmann average of the vibronic spectra of the stable conformers, and includes the contribution of structures that sample all the accessible conformational space. It is applied to the simulation of the ECD spectrum of (S)-2,2,2-trifluoroanthrylethanol, a molecule with easily interconvertible conformers, whose spectrum exhibits a pattern of alternating positive and negative vibronic peaks. Results are in very good agreement with experiment and show that spectra averaged over all the sampled conformational space can deviate significantly from the simple average of the contributions of the stable conformers. The present mixed quantum/classical method is able to capture the effect of the nonlinear dependence of the rotatory strength on the molecular structure and of the anharmonic couplings among the modes responsible for molecular flexibility. Despite its computational cost, the procedure is still affordable and promises to be useful in all cases where the ECD shape arises from a subtle balance between vibronic effects and conformational variety. © 2018 Wiley Periodicals, Inc.

Molecular dynamics simulations of certain RGD-based peptides from Kistrin provide insight into the higher activity of REI-RGD34 protein at higher temperature.

PubMed

Upadhyay, Sanjay K

2014-05-01

To determine the bioactive conformation required to bind with receptor aIIbb3, the peptide sequence RIPRGDMP from Kistrin was inserted into CDR 1 loop region of REI protein, resulting in REI-RGD34. The activity of REI-RGD34 was observed to increase at higher temperature towards the receptor aIIbb3. It could be justified in either way: the modified complex forces the restricted peptide to adapt bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. Here, we model the conformational preference of RGD sequence in RIPRGDMP at 25 and 42 °C using multiple MD simulations. Further, we model the peptide sequence RGD, PRGD and PRGDMP from kistrin to observe the effect of flanking residues on conformational sampling of RGD. The presence of flanking residues around RGD peptide greatly influenced the conformational sampling. A transition from bend to turn conformation was observed for RGD sequence at 42 °C. The turn conformation shows pharmacophoric parameters required to recognize the receptor aIIbb3. Thus, the temperaturedependent activity of RIPRGDMP when inserted into the loop region of REI can be explained by the presence of the turn conformation. This study will help in designing potential antagonist for the receptor aIIbb3.

Temporal trends of Dechlorane Plus in air and precipitation around the North American Great Lakes.

PubMed

Olukunle, Olubiyi I; Lehman, Daniel C; Salamova, Amina; Venier, Marta; Hites, Ronald A

2018-06-13

Dechlorane Plus (DP) is a chlorinated flame retardant manufactured only in Niagara Falls, New York and in Huai'an, China. To determine if the environmental levels of this compound were changing significantly, we measured the long-term temporal trends of its concentrations near the Great Lakes between 2005 and 2015 using air (vapor + particle phase) samples (N = 1047) and precipitation samples (N = 449). We used a multiple linear regression model of DP concentrations to isolate the variabilities due to sampling date and population near the sampling site. The results show that the total DP concentrations in precipitation varied seasonally, maximizing on January 18, but the concentrations in the vapor + particle phase did not show seasonal variations. Vapor + particle phase DP levels were relatively high in Cleveland, and precipitation DP levels were relatively high at Point Petre. DP's concentrations in neither phase were changing as a function of sampling date, indicating that the input of this compound into the environment is continuing, presumably because its use and production are not regulated. Based on the ratio of the anti conformer relative to the total of the two conformer concentrations, we suggest that the syn conformer is somewhat more environmentally stable than the anti conformer. Copyright © 2018 Elsevier B.V. All rights reserved.

Impact of Educational Activities in Reducing Pre-Analytical Laboratory Errors

PubMed Central

Al-Ghaithi, Hamed; Pathare, Anil; Al-Mamari, Sahimah; Villacrucis, Rodrigo; Fawaz, Naglaa; Alkindi, Salam

2017-01-01

Objectives Pre-analytic errors during diagnostic laboratory investigations can lead to increased patient morbidity and mortality. This study aimed to ascertain the effect of educational nursing activities on the incidence of pre-analytical errors resulting in non-conforming blood samples. Methods This study was conducted between January 2008 and December 2015. All specimens received at the Haematology Laboratory of the Sultan Qaboos University Hospital, Muscat, Oman, during this period were prospectively collected and analysed. Similar data from 2007 were collected retrospectively and used as a baseline for comparison. Non-conforming samples were defined as either clotted samples, haemolysed samples, use of the wrong anticoagulant, insufficient quantities of blood collected, incorrect/lack of labelling on a sample or lack of delivery of a sample in spite of a sample request. From 2008 onwards, multiple educational training activities directed at the hospital nursing staff and nursing students primarily responsible for blood collection were implemented on a regular basis. Results After initiating corrective measures in 2008, a progressive reduction in the percentage of non-conforming samples was observed from 2009 onwards. Despite a 127.84% increase in the total number of specimens received, there was a significant reduction in non-conforming samples from 0.29% in 2007 to 0.07% in 2015, resulting in an improvement of 75.86% (P <0.050). In particular, specimen identification errors decreased by 0.056%, with a 96.55% improvement. Conclusion Targeted educational activities directed primarily towards hospital nursing staff had a positive impact on the quality of laboratory specimens by significantly reducing pre-analytical errors. PMID:29062553

Impact of Educational Activities in Reducing Pre-Analytical Laboratory Errors: A quality initiative.

PubMed

Al-Ghaithi, Hamed; Pathare, Anil; Al-Mamari, Sahimah; Villacrucis, Rodrigo; Fawaz, Naglaa; Alkindi, Salam

2017-08-01

Pre-analytic errors during diagnostic laboratory investigations can lead to increased patient morbidity and mortality. This study aimed to ascertain the effect of educational nursing activities on the incidence of pre-analytical errors resulting in non-conforming blood samples. This study was conducted between January 2008 and December 2015. All specimens received at the Haematology Laboratory of the Sultan Qaboos University Hospital, Muscat, Oman, during this period were prospectively collected and analysed. Similar data from 2007 were collected retrospectively and used as a baseline for comparison. Non-conforming samples were defined as either clotted samples, haemolysed samples, use of the wrong anticoagulant, insufficient quantities of blood collected, incorrect/lack of labelling on a sample or lack of delivery of a sample in spite of a sample request. From 2008 onwards, multiple educational training activities directed at the hospital nursing staff and nursing students primarily responsible for blood collection were implemented on a regular basis. After initiating corrective measures in 2008, a progressive reduction in the percentage of non-conforming samples was observed from 2009 onwards. Despite a 127.84% increase in the total number of specimens received, there was a significant reduction in non-conforming samples from 0.29% in 2007 to 0.07% in 2015, resulting in an improvement of 75.86% ( P <0.050). In particular, specimen identification errors decreased by 0.056%, with a 96.55% improvement. Targeted educational activities directed primarily towards hospital nursing staff had a positive impact on the quality of laboratory specimens by significantly reducing pre-analytical errors.

Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method.

PubMed

Molloy, Kevin; Shehu, Amarda

2013-01-01

Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting structural states that are significantly different. A detailed analysis on the depth and breadth of the tree suggests that a soft global bias over the progress coordinate enhances sampling and results in higher path diversity. The explicit geometric projection layer that biases the exploration away from over-sampled regions further increases coverage, often improving proximity to the goal by forcing the exploration to find new paths. The reactive temperature scheme is shown effective in increasing path diversity, particularly in difficult structural transitions with known high-energy barriers.

The Role of Molecular Dynamics Potential of Mean Force Calculations in the Investigation of Enzyme Catalysis.

PubMed

Yang, Y; Pan, L; Lightstone, F C; Merz, K M

2016-01-01

The potential of mean force simulations, widely applied in Monte Carlo or molecular dynamics simulations, are useful tools to examine the free energy variation as a function of one or more specific reaction coordinate(s) for a given system. Implementation of the potential of mean force in the simulations of biological processes, such as enzyme catalysis, can help overcome the difficulties of sampling specific regions on the energy landscape and provide useful insights to understand the catalytic mechanism. The potential of mean force simulations usually require many, possibly parallelizable, short simulations instead of a few extremely long simulations and, therefore, are fairly manageable for most research facilities. In this chapter, we provide detailed protocols for applying the potential of mean force simulations to investigate enzymatic mechanisms for several different enzyme systems. © 2016 Elsevier Inc. All rights reserved.

Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

PubMed Central

Wang, Zhen; Antoniou, Dimitri; Schwartz, Steven D.; Schramm, Vern L.

2016-01-01

Escherichia coli dihydrofolate reductase (ecDHFR) is used to study fundamental principles of enzyme catalysis. It remains controversial whether fast protein motions are coupled to the hydride transfer catalyzed by ecDHFR. Previous studies with heavy ecDHFR proteins labeled with 13C, 15N, and nonexchangeable 2H reported enzyme mass-dependent hydride transfer kinetics for ecDHFR. Here, we report refined experimental and computational studies to establish that hydride transfer is independent of protein mass. Instead, we found the rate constant for substrate dissociation to be faster for heavy DHFR. Previously reported kinetic differences between light and heavy DHFRs likely arise from kinetic steps other than the chemical step. This study confirms that fast (femtosecond to picosecond) protein motions in ecDHFR are not coupled to hydride transfer and provides an integrative computational and experimental approach to resolve fast dynamics coupled to chemical steps in enzyme catalysis. PMID:26652185

Polarization Effects on the Cellulose Dissolution in Ionic Liquids: Molecular Dynamics Simulations with Polarization Model and Integrated Tempering Enhanced Sampling Method.

PubMed

Kan, Zigui; Zhu, Qiang; Yang, Lijiang; Huang, Zhixiong; Jin, Biaobing; Ma, Jing

2017-05-04

Conformation of cellulose with various degree of polymerization of n = 1-12 in ionic liquid 1,3-dimethylimidazolium chloride ([C 1 mim]Cl) and the intermolecular interaction between them was studied by means of molecular dynamics (MD) simulations with fixed-charge and charge variable polarizable force fields, respectively. The integrated tempering enhanced sampling method was also employed in the simulations in order to improve the sampling efficiency. Cellulose undergoes significant conformational changes from a gaseous right-hand helical twist along the long axis to a flexible conformation in ionic liquid. The intermolecular interactions between cellulose and ionic liquid were studied by both infrared spectrum measurements and theoretical simulations. Designated by their puckering parameters, the pyranose rings of cellulose oligomers are mainly arranged in a chair conformation. With the increase in the degree of polymerization of cellulose, the boat and skew-boat conformations of cellulose appear in the MD simulations, especially in the simulations with polarization model. The number and population of hydrogen bonds between the cellulose and the chloride anions show that chloride anion is prone to form HBs whenever it approaches the hydroxyl groups of cellulose and, thus, each hydroxyl group is fully hydrogen bonded to the chloride anion. MD simulations with polarization model presented more abundant conformations than that with nonpolarization model. The application of the enhanced sampling method further enlarged the conformational spaces that could be visited by facilitating the system escaping from the local minima. It was found that the electrostatics interactions between the cellulose and ionic liquid contribute more to the total interaction energies than the van der Waals interactions. Although the interaction energy between the cellulose and anion is about 2.9 times that between the cellulose and cation, the role of cation is non-negligible. In contrast, the interaction energy between the cellulose and water is too weak to dissolve cellulose in water.

Constrained proper sampling of conformations of transition state ensemble of protein folding

PubMed Central

Lin, Ming; Zhang, Jian; Lu, Hsiao-Mei; Chen, Rong; Liang, Jie

2011-01-01

Characterizing the conformations of protein in the transition state ensemble (TSE) is important for studying protein folding. A promising approach pioneered by Vendruscolo [Nature (London) 409, 641 (2001)] to study TSE is to generate conformations that satisfy all constraints imposed by the experimentally measured ϕ values that provide information about the native likeness of the transition states. Faísca [J. Chem. Phys. 129, 095108 (2008)] generated conformations of TSE based on the criterion that, starting from a TS conformation, the probabilities of folding and unfolding are about equal through Markov Chain Monte Carlo (MCMC) simulations. In this study, we use the technique of constrained sequential Monte Carlo method [Lin , J. Chem. Phys. 129, 094101 (2008); Zhang Proteins 66, 61 (2007)] to generate TSE conformations of acylphosphatase of 98 residues that satisfy the ϕ-value constraints, as well as the criterion that each conformation has a folding probability of 0.5 by Monte Carlo simulations. We adopt a two stage process and first generate 5000 contact maps satisfying the ϕ-value constraints. Each contact map is then used to generate 1000 properly weighted conformations. After clustering similar conformations, we obtain a set of properly weighted samples of 4185 candidate clusters. Representative conformation of each of these cluster is then selected and 50 runs of Markov chain Monte Carlo (MCMC) simulation are carried using a regrowth move set. We then select a subset of 1501 conformations that have equal probabilities to fold and to unfold as the set of TSE. These 1501 samples characterize well the distribution of transition state ensemble conformations of acylphosphatase. Compared with previous studies, our approach can access much wider conformational space and can objectively generate conformations that satisfy the ϕ-value constraints and the criterion of 0.5 folding probability without bias. In contrast to previous studies, our results show that transition state conformations are very diverse and are far from nativelike when measured in cartesian root-mean-square deviation (cRMSD): the average cRMSD between TSE conformations and the native structure is 9.4 Å  for this short protein, instead of 6 Å reported in previous studies. In addition, we found that the average fraction of native contacts in the TSE is 0.37, with enrichment in native-like β-sheets and a shortage of long range contacts, suggesting such contacts form at a later stage of folding. We further calculate the first passage time of folding of TSE conformations through calculation of physical time associated with the regrowth moves in MCMC simulation through mapping such moves to a Markovian state model, whose transition time was obtained by Langevin dynamics simulations. Our results indicate that despite the large structural diversity of the TSE, they are characterized by similar folding time. Our approach is general and can be used to study TSE in other macromolecules. PMID:21341875

Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

PubMed

Schwaiger, K; Wimmer, M; Huber-Schlenstedt, R; Fehlings, K; Hölzel, C S; Bauer, J

2012-01-01

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Asymmetric Aldol Additions: A Guided-Inquiry Laboratory Activity on Catalysis

ERIC Educational Resources Information Center

King, Jorge H. Torres; Wang, Hong; Yezierski, Ellen J.

2018-01-01

Despite the importance of asymmetric catalysis in both the pharmaceutical and commodity chemicals industries, asymmetric catalysis is under-represented in undergraduate chemistry laboratory curricula. A novel guided-inquiry experiment based on the asymmetric aldol addition was developed. Students conduct lab work to compare the effectiveness of…

Advances in Nucleophilic Phosphine Catalysis of Alkenes, Allenes, Alkynes, and MBHADs

PubMed Central

Fan, Yi Chiao

2014-01-01

In nucleophilic phosphine catalysis, tertiary phosphines undergo conjugate additions to activated carbon–carbon multiple bonds to form β-phosphonium enolates, β-phosphonium dienolates, β-phosphonium enoates, and vinyl phosphonium ylides as intermediates. When these reactive zwitterionic species react with nucleophiles and electrophiles, they may generate carbo- and heterocycles with multifarious molecular architectures. This Article describes the reactivities of these phosphonium zwitterions, the applications of phosphine catalysis in the syntheses of biologically active compounds and natural products, and recent developments in the enantioselective phosphine catalysis. PMID:24196409

Magnetic catalysis and inverse magnetic catalysis in (2 +1 )-dimensional gauge theories from holographic models

NASA Astrophysics Data System (ADS)

Rodrigues, Diego M.; Capossoli, Eduardo Folco; Boschi-Filho, Henrique

2018-06-01

We study the deconfinement phase transition in (2 +1 )-dimensional holographic S U (N ) gauge theories in the presence of an external magnetic field from the holographic hard and soft wall models. We obtain exact solutions for the critical temperature of the deconfinement transition for any range of magnetic field. As a consequence, we find a critical magnetic field (Bc), in which the critical temperature (Tc) vanishes; for B Bc we have a magnetic catalysis.

Modeling the effect of pathogenic mutations on the conformational landscape of protein kinases.

PubMed

Saladino, Giorgio; Gervasio, Francesco Luigi

2016-04-01

Most proteins assume different conformations to perform their cellular functions. This conformational dynamics is physiologically regulated by binding events and post-translational modifications, but can also be affected by pathogenic mutations. Atomistic molecular dynamics simulations complemented by enhanced sampling approaches are increasingly used to probe the effect of mutations on the conformational dynamics and on the underlying conformational free energy landscape of proteins. In this short review we discuss recent successful examples of simulations used to understand the molecular mechanism underlying the deregulation of physiological conformational dynamics due to non-synonymous single point mutations. Our examples are mostly drawn from the protein kinase family. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruski, Marek; Sadow, Aaron D.; Slowing, Igor I.

Catalysis research at the U.S. Department of Energy's (DOE's) National Laboratories covers a wide range of research topics in heterogeneous catalysis, homogeneous/molecular catalysis, biocatalysis, electrocatalysis, and surface science. Since much of the work at National Laboratories is funded by DOE, the research is largely focused on addressing DOE's mission to ensure America's security and prosperity by addressing its energy, environmental, and nuclear challenges through transformative science and technology solutions. The catalysis research carried out at the DOE National Laboratories ranges from very fundamental catalysis science, funded by DOE's Office of Basic Energy Sciences (BES), to applied research and development (R&D)more » in areas such as biomass conversion to fuels and chemicals, fuel cells, and vehicle emission control with primary funding from DOE's Office of Energy Efficiency and Renewable Energy.« less

Coupling chemical and biological catalysis: a flexible paradigm for producing biobased chemicals.

PubMed

Schwartz, Thomas J; Shanks, Brent H; Dumesic, James A

2016-04-01

Advances in metabolic engineering have allowed for the development of new biological catalysts capable of selectively de-functionalizing biomass to yield platform molecules that can be upgraded to biobased chemicals using high efficiency continuous processing allowed by heterogeneous chemical catalysis. Coupling these disciplines overcomes the difficulties of selectively activating COH bonds by heterogeneous chemical catalysis and producing petroleum analogues by biological catalysis. We show that carboxylic acids, pyrones, and alcohols are highly flexible platforms that can be used to produce biobased chemicals by this approach. More generally, we suggest that molecules with three distinct functionalities may represent a practical upper limit on the extent of functionality present in the platform molecules that serve as the bridge between biological and chemical catalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanochemical depolymerization of inulin.

PubMed

Xing, Haoran; Yaylayan, Varoujan A

2018-05-02

Although chemical reactions driven by mechanical force is emerging as a promising tool in the field of physical sciences, its applications in the area of food sciences are not reported. In this paper, we propose ball milling as an efficient tool for the controlled generation of fructooligosaccharide (FOS) mixtures from inulin with a degree of polymerization (dp) ranging between 4 and 7. The addition of catalytic amounts of AlCl 3 together with ball milling (30 min, at 30 Hz) generated mixtures rich in dehydrated disaccharides such as di-D-fructose dianhydrides. Based on anion exchange chromatography in conjunction with ESI/qTOF/MS/MS analysis, catalysis increased the overall content of mono-, di-, and tri-saccharides by around 30 fold compared to un-catalyzed milling. In addition, dialysis results of the untreated and treated samples have indicated that under catalysis the percent of depolymerization (dp < 12) reached 73.4% from the starting value of 27.6% in the untreated sample. Both processes resulted in mixtures of prebiotic value. The use of mechanical energy may be suitable for a fast, cost-efficient and green conversion of inulin to value-added food ingredients. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

PubMed Central

Allison, Kim; Robbins, Thomas; Lyubimov, Artem Y.; Uervirojnangkoorn, Monarin; Brunger, Axel T.; Khosla, Chaitan; DeMirci, Hasan; McPhillips, Scott E.; Hollenbeck, Michael; Soltis, Michael; Cohen, Aina E.

2017-01-01

The crystal structure of the trans-acyltrans-ferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed. PMID:28832129

Enhanced Conformational Sampling Using Replica Exchange with Collective-Variable Tempering

PubMed Central

2015-01-01

The computational study of conformational transitions in RNA and proteins with atomistic molecular dynamics often requires suitable enhanced sampling techniques. We here introduce a novel method where concurrent metadynamics are integrated in a Hamiltonian replica-exchange scheme. The ladder of replicas is built with different strengths of the bias potential exploiting the tunability of well-tempered metadynamics. Using this method, free-energy barriers of individual collective variables are significantly reduced compared with simple force-field scaling. The introduced methodology is flexible and allows adaptive bias potentials to be self-consistently constructed for a large number of simple collective variables, such as distances and dihedral angles. The method is tested on alanine dipeptide and applied to the difficult problem of conformational sampling in a tetranucleotide. PMID:25838811

myPresto/omegagene: a GPU-accelerated molecular dynamics simulator tailored for enhanced conformational sampling methods with a non-Ewald electrostatic scheme.

PubMed

Kasahara, Kota; Ma, Benson; Goto, Kota; Dasgupta, Bhaskar; Higo, Junichi; Fukuda, Ikuo; Mashimo, Tadaaki; Akiyama, Yutaka; Nakamura, Haruki

2016-01-01

Molecular dynamics (MD) is a promising computational approach to investigate dynamical behavior of molecular systems at the atomic level. Here, we present a new MD simulation engine named "myPresto/omegagene" that is tailored for enhanced conformational sampling methods with a non-Ewald electrostatic potential scheme. Our enhanced conformational sampling methods, e.g. , the virtual-system-coupled multi-canonical MD (V-McMD) method, replace a multi-process parallelized run with multiple independent runs to avoid inter-node communication overhead. In addition, adopting the non-Ewald-based zero-multipole summation method (ZMM) makes it possible to eliminate the Fourier space calculations altogether. The combination of these state-of-the-art techniques realizes efficient and accurate calculations of the conformational ensemble at an equilibrium state. By taking these advantages, myPresto/omegagene is specialized for the single process execution with Graphics Processing Unit (GPU). We performed benchmark simulations for the 20-mer peptide, Trp-cage, with explicit solvent. One of the most thermodynamically stable conformations generated by the V-McMD simulation is very similar to an experimentally solved native conformation. Furthermore, the computation speed is four-times faster than that of our previous simulation engine, myPresto/psygene-G. The new simulator, myPresto/omegagene, is freely available at the following URLs: http://www.protein.osaka-u.ac.jp/rcsfp/pi/omegagene/ and http://presto.protein.osaka-u.ac.jp/myPresto4/.

The mechanism of montmorillonite catalysis in RNA synthesis

NASA Astrophysics Data System (ADS)

Joshi, Prakash

The formation of complex prebiotic molecules on the early Earth is likely to have involved a component of mineral catalysis. Amongst the variety of clay minerals that have been investigated by us for their ability to catalyze the formation of RNA oligomers is montmorillonite. These are 2:1 layer silicates that have a wide range of chemical compositions [(Na,Ca)0.33(Al,Fe,Mg)2(Si,Al)4O10(OH)2.nH2O]. They are commonly produced by the weathering of silicic volcanic ashes to form Bentonite. Once formed, montmorillonites gradually transform to Illites at a modest pressure and temperature. Of the many samples of montmorillonite that we have experimentally examined, a selected subset has been observed to be catalytic for RNA synthesis (Joshi et. al., 2009; Aldersley et al., 2011). Those that have been observed to be excellent catalysts come from a restricted range of elemental compositions. The recent identification of phyllosilicates including montmorillonite on Mars (Bishop et al., 2008) raises the possibility that such processes may have taken place there too. The extent of catalysis depended not only upon the magnitude of the negative charge on the montmorillonite lattice and the number of cations associated with it, but also on the pH at which the reaction is promoted. The isotherm and catalysis studies were extended to provide binding information and catalytic outcomes over a wide pH range. When cations in raw montmorillonite are completely replaced by sodium ions, the resulting Na+-montmorillonite does not catalyze oligomer formation because the ions saturate the interlayer between the platelets of montmorillonite, which blocks the binding of the activated monomers. Acid washed montmorillonite titrated to pH 6-8 with alkali metal ions, serves as the model catalyst for this RNA synthesis (Aldersley et. al., 2011). The optimal binding occurred in the region of maximal oligomer formation. X-ray diffraction studies revealed changes in layer separations of montmorillonite as reaction occurs. The application of the Scherer equation to the X-ray diffraction data showed differences in domain size. Modeling of the size of the activated nucleotide monomers and the charge on the montmorillonite surface provided an interpretation of how these factors influence adsorption. This research provides a basis for further understanding of the physical processes in the mechanism of this catalysis in prebiotic reactions. This research was supported by NASA Astrobiology Institute Grant NNA09DA80A. References: Aldersley, M.F., Joshi, P.C., Price, J.D., Ferris, J.P. The role of montmorillonite in its catalysis of RNA synthesis. Appl. Clay Sci. 54,1-14, 2011. Bishop, J.L., Dobrea, E.J.N., Mckeown, N.K., Parenta, M. Phyllos- ilicate diversity and past aqueous activity revealed at Mawrth Vallis, Mars. Science 321, 830-833, 2008. Joshi, P.C., Aldersley, M.F., Delano, J.W., Ferris, J.P., Mechanism of montmorillonite catalysis in the formation of RNA oligomers, J. Am. Chem. Soc., 131, 13369-13374, 2009.

Heterogeneous Catalysis with Renewed Attention: Principles, Theories, and Concepts

ERIC Educational Resources Information Center

Dumeignil, Franck; Paul, Jean-Francois; Paul, Sebastien

2017-01-01

With the development of a strong bioeconomy sector related to the creation of next-generation biorefineries, heterogeneous catalysis is receiving renewed attention. Indeed, catalysis is at the core of biorefinery design, and many new catalysts and catalytic processes are being developed. On the one hand, they are based on knowledge acquired during…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Jingguang; Frenkel, Anatoly; Rodriguez, Jose

Synchrotron spectroscopies offer unique advantages over conventional techniques, including higher detection sensitivity and molecular specificity, faster detection rate, and more in-depth information regarding the structural, electronic and catalytic properties under in-situ reaction conditions. Despite these advantages, synchrotron techniques are often underutilized or unexplored by the catalysis community due to various perceived and real barriers, which will be addressed in the current proposal. Since its establishment in 2005, the Synchrotron Catalysis Consortium (SCC) has coordinated significant efforts to promote the utilization of cutting-edge catalytic research under in-situ conditions. The purpose of the current renewal proposal is aimed to provide assistance, andmore » to develop new sciences/techniques, for the catalysis community through the following concerted efforts: Coordinating the implementation of a suite of beamlines for catalysis studies at the new NSLS-II synchrotron source; Providing assistance and coordination for catalysis users at an SSRL catalysis beamline during the initial period of NSLS to NSLS II transition; Designing in-situ reactors for a variety of catalytic and electrocatalytic studies; Assisting experimental set-up and data analysis by a dedicated research scientist; Offering training courses and help sessions by the PIs and co-PIs.« less

Solvent, Temperature And Concentration Effects on the Optical Activity of Chiral FIVE-And-SIX Membered Ring Ketones Conformers

NASA Astrophysics Data System (ADS)

Al-Basheer, Watheq

2017-06-01

Chiral five-and-six membered ring ketones are important molecules that are found in many biological systems and can exist in many possible conformers. In this talk, experimental and computational investigation of solvent, temperature and concentration effects on the circular dichroism (CD) and optical rotation (OR) of (R)-3 -methylcyclohexanone (R3MCH), (R)-3-methylcyclopentanone (R3MCP) and carvone conformers will be discussed. CD and OR measurements of these ketones gaseous samples and in ten common solvents of wide polarity range for different concentrations and sample temperatures were recorded and related to molecular conformation. Density functional theoretical calculations were performed using Gaussian09 at B3LYP functions with aug-cc-pVDZ level of theory. Also, CD and OR spectra for the optimized geometries of the ketones dominant conformers were computed over the ultraviolet and visible region in the gas phase as well as in ten solvents of varying polarity range, and under the umbrella of the polarizable continuum model (PCM). By comparing theoretical and experimental results, few thermodynamic parameters were deduced for the individual equatorial and axial conformers of each molecule in gas phase and in solvation.

Metadynamics in the conformational space nonlinearly dimensionally reduced by Isomap

NASA Astrophysics Data System (ADS)

Spiwok, Vojtěch; Králová, Blanka

2011-12-01

Atomic motions in molecules are not linear. This infers that nonlinear dimensionality reduction methods can outperform linear ones in analysis of collective atomic motions. In addition, nonlinear collective motions can be used as potentially efficient guides for biased simulation techniques. Here we present a simulation with a bias potential acting in the directions of collective motions determined by a nonlinear dimensionality reduction method. Ad hoc generated conformations of trans,trans-1,2,4-trifluorocyclooctane were analyzed by Isomap method to map these 72-dimensional coordinates to three dimensions, as described by Brown and co-workers [J. Chem. Phys. 129, 064118 (2008)]. Metadynamics employing the three-dimensional embeddings as collective variables was applied to explore all relevant conformations of the studied system and to calculate its conformational free energy surface. The method sampled all relevant conformations (boat, boat-chair, and crown) and corresponding transition structures inaccessible by an unbiased simulation. This scheme allows to use essentially any parameter of the system as a collective variable in biased simulations. Moreover, the scheme we used for mapping out-of-sample conformations from the 72D to 3D space can be used as a general purpose mapping for dimensionality reduction, beyond the context of molecular modeling.

Conformational changes in matrix-isolated 6-methoxyindole: Effects of the thermal and infrared light excitations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopes Jesus, A. J.; CQC, Faculty of Pharmacy, University of Coimbra, 3004-295 Coimbra; Reva, I., E-mail: reva@qui.uc.pt

2016-03-28

Conformational changes induced thermally or upon infrared excitation of matrix-isolated 6-methoxyindole were investigated. Narrowband near-infrared excitation of the first overtone of the N–H stretching vibration of each one of the two identified conformers is found to induce a selective large-scale conversion of the pumped conformer into the other one. This easily controllable bidirectional process consists in the intramolecular reorientation of the methoxy group and allowed a full assignment of the infrared spectra of the two conformers. Matrices with different conformational compositions prepared by narrow-band irradiations were subsequently used to investigate the effects of both thermal and broadband infrared excitations onmore » the conformational mixtures. Particular attention is given to the influence of the matrix medium (Ar vs. Xe) and conformational effects of exposition of the sample to the spectrometer light source during the measurements.« less

Unique aspects of the structure and dynamics of elementary Iβ cellulose microfibrils revealed by computational simulations.

PubMed

Oehme, Daniel P; Downton, Matthew T; Doblin, Monika S; Wagner, John; Gidley, Michael J; Bacic, Antony

2015-05-01

The question of how many chains an elementary cellulose microfibril contains is critical to understanding the molecular mechanism(s) of cellulose biosynthesis and regulation. Given the hexagonal nature of the cellulose synthase rosette, it is assumed that the number of chains must be a multiple of six. We present molecular dynamics simulations on three different models of Iβ cellulose microfibrils, 18, 24, and 36 chains, to investigate their structure and dynamics in a hydrated environment. The 36-chain model stays in a conformational space that is very similar to the initial crystalline phase, while the 18- and 24-chain models sample a conformational space different from the crystalline structure yet similar to conformations observed in recent high-temperature molecular dynamics simulations. Major differences in the conformations sampled between the different models result from changes to the tilt of chains in different layers, specifically a second stage of tilt, increased rotation about the O2-C2 dihedral, and a greater sampling of non-TG exocyclic conformations, particularly the GG conformation in center layers and GT conformation in solvent-exposed exocyclic groups. With a reinterpretation of nuclear magnetic resonance data, specifically for contributions made to the C6 peak, data from the simulations suggest that the 18- and 24-chain structures are more viable models for an elementary cellulose microfibril, which also correlates with recent scattering and diffraction experimental data. These data inform biochemical and molecular studies that must explain how a six-particle cellulose synthase complex rosette synthesizes microfibrils likely comprised of either 18 or 24 chains. © 2015 American Society of Plant Biologists. All Rights Reserved.

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

PubMed Central

Perroud, Thomas D.; Bokoch, Michael P.; Zare, Richard N.

2005-01-01

We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pH-induced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0–9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5–10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry. PMID:16314563

Using simulation to interpret experimental data in terms of protein conformational ensembles.

PubMed

Allison, Jane R

2017-04-01

In their biological environment, proteins are dynamic molecules, necessitating an ensemble structural description. Molecular dynamics simulations and solution-state experiments provide complimentary information in the form of atomically detailed coordinates and averaged or distributions of structural properties or related quantities. Recently, increases in the temporal and spatial scale of conformational sampling and comparison of the more diverse conformational ensembles thus generated have revealed the importance of sampling rare events. Excitingly, new methods based on maximum entropy and Bayesian inference are promising to provide a statistically sound mechanism for combining experimental data with molecular dynamics simulations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

PubMed

Chen, Mengbin; Chou, Wayne K W; Al-Lami, Naeemah; Faraldos, Juan A; Allemann, Rudolf K; Cane, David E; Christianson, David W

2016-05-24

Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility.

PubMed

Crofts, A R; Guergova-Kuras, M; Huang, L; Kuras, R; Zhang, Z; Berry, E A

1999-11-30

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

PubMed Central

Lustig, B; Lin, N H; Smith, S M; Jernigan, R L; Jeang, K T

1995-01-01

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason. Images PMID:7567466

Insight into the role of Mg2+ in hammerhead ribozyme catalysis from X-ray crystallography and molecular dynamics simulation

PubMed Central

Lee, Tai-Sung; López, Carlos Silva; Martick, Monika; Scott, William G.; York, Darrin M.

2008-01-01

Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg2+ ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg2+ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg2+ spends significant time closely associated with the 2′OH of G8, but remains fairly distant from the leaving group O5′ position. In the early TS mimic simulation, where the nucleophilic O2′ and leaving group O5′ are equidistant from the phosphorus, the Mg2+ ion remains tightly coordinated to the 2′OH of G8, but is positioned closer to the O5′ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg2+ ion undergoes a transition whereby the coordination with the 2′OH of G8 is replace by the leaving group O5′ that has developed significant charge. At the same time, the 2′OH of G8 forms a hydrogen bond with the leaving group O5′ and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg2+ ion in catalysis that is consistent with both crystallographic and biochemical data. PMID:19079784

Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide

PubMed Central

Li, Chun-Yang; Wei, Tian-Di; Zhang, Sheng-Hui; Chen, Xiu-Lan; Gao, Xiang; Wang, Peng; Xie, Bin-Bin; Su, Hai-Nan; Qin, Qi-Long; Zhang, Xi-Ying; Yu, Juan; Zhang, Hong-Hai; Zhou, Bai-Cheng; Yang, Gui-Peng; Zhang, Yu-Zhong

2014-01-01

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI_1157. The structures of DddQ bound to an inhibitory molecule 2-(N-morpholino)ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure and contains a Zn2+ ion and six highly conserved hydrophilic residues (Tyr120, His123, His125, Glu129, Tyr131, and His163) in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between “open” and “closed” states, serving as a gate for DMSP entry. We also propose a molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally important biogeochemical reaction. PMID:24395783

Heteromultimetallic catalysis for sustainable organic syntheses.

PubMed

Lorion, Mélanie M; Maindan, Karan; Kapdi, Anant R; Ackermann, Lutz

2017-12-07

Fully complementary bimetallic catalysis has been identified as an increasingly powerful tool for molecular transformations, which was largely inspired by early examples of sequential catalytic transformations. Thus, energy-efficient one-pot reactions involving different metal catalysts orchestrated in concert constitute an attractive alternative to multi-step protocols, with major recent progress through the elegant ligand design in heterobimetallic catalysis as well as sustainable photo-induced C-H transformations, among others. This review provides a critical assessment of the state of the art in heterobimetallic catalysis for sustainable organic syntheses (SOS), highlighting key advances and representative examples until summer 2017.

Geometric Restraint Drives On- and Off-pathway Catalysis by the Escherichia coli Menaquinol:Fumarate Reductase*

PubMed Central

Tomasiak, Thomas M.; Archuleta, Tara L.; Andréll, Juni; Luna-Chávez, César; Davis, Tyler A.; Sarwar, Maruf; Ham, Amy J.; McDonald, W. Hayes; Yankovskaya, Victoria; Stern, Harry A.; Johnston, Jeffrey N.; Maklashina, Elena; Cecchini, Gary; Iverson, Tina M.

2011-01-01

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2–C3 double bond of activated fumarate parallel to the C(4a)–N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR. PMID:21098488

Geometric Restraint Drives On- and Off-pathway Catalysis by the Escherichia coli Menaquinol:Fumarate Reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomasiak, Thomas M.; Archuleta, Tara L.; Andréll, Juni

2012-01-05

Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of themore » C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.« less

Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology.

PubMed

Makam, Pandeeswar; Gazit, Ehud

2018-05-21

Molecular self-assembly is a ubiquitous process in nature and central to bottom-up nanotechnology. In particular, the organization of peptide building blocks into ordered supramolecular structures has gained much interest due to the unique properties of the products, including biocompatibility, chemical and structural diversity, robustness and ease of large-scale synthesis. In addition, peptides, as short as dipeptides, contain all the molecular information needed to spontaneously form well-ordered structures at both the nano- and the micro-scale. Therefore, peptide supramolecular assembly has been effectively utilized to produce novel materials with tailored properties for various applications in the fields of material science, engineering, medicine, and biology. To further expand the conformational space of peptide assemblies in terms of structural and functional complexity, multicomponent (two or more) peptide supramolecular co-assembly has recently evolved as a promising extended approach, similar to the structural diversity of natural sequence-defined biopolymers (proteins) as well as of synthetic covalent co-polymers. The use of this methodology was recently demonstrated in various applications, such as nanostructure physical dimension control, the creation of non-canonical complex topologies, mechanical strength modulation, the design of light harvesting soft materials, fabrication of electrically conducting devices, induced fluorescence, enzymatic catalysis and tissue engineering. In light of these significant advancements in the field of peptide supramolecular co-assembly in the last few years, in this tutorial review, we provide an updated overview and future prospects of this emerging subject.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.

PubMed

Wan, Qun; Parks, Jerry M; Hanson, B Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E; Graham, David E; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

2015-10-06

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD=pH+0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

PubMed Central

Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

2015-01-01

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen. PMID:26392527

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

PubMed Central

Huang, Xuhui; Wang, Dong; Weiss, Dahlia R.; Bushnell, David A.; Kornberg, Roger D.; Levitt, Michael

2010-01-01

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the “closed” conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. PMID:20798057

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Qun; Parks, Jerry M.; Hanson, B. Leif

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. Themore » general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.« less

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

DOE PAGES

Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; ...

2015-09-21

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. Themore » general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.« less

Part I: RNA hydrolysis catalyzed by imidazole compounds. Part II. Hydrophobic acceleration of reactions and mimics of thiamin-dependent enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kool, E.T.

1988-01-01

Catalysts modeled after the active site groups of the enzyme Ribonuclease A were synthesized and tested for catalysis of the hydrolysis of poly(rU), using a quantitative assay. The most effective of all the catalysts is N,N{prime}-bis-imidazolylmethane, which gave a four-fold rate enhancement as compared to N-methyl-imidazole. The structure/activity relationships are discussed in light of the ribonuclease mechanism. Also examined were reactions catalyzed by the coenzyme thiamine. In an investigation of the effects of restricting conformational freedom, a thiazolium salt was attached in two positions to {beta}-cyclodextrin. Since the catalyst gave about the same rate for tritium exchange from benzaldehyde asmore » singly-attached catalysts, we surmise that any rate enhancement due to the restriction of bond rotations has been lost by forcing the structure into less productive conformations. The benzoin condensation catalyzed by cyanide was also investigated. The reaction was shown to be faster in water than in most organic solvents. Kinetic salt effects and the effects of added {beta}- and {gamma}-cyclodextrin were measured in water; salting-out ions and {gamma}-cyclodextrin increase the rate, while salting-in ions and {beta}-cyclodextrin decrease it. Negative salt effects were observed in formamide, ethylene glycol, and DMSO. All these media effects are discussed in relation to the compact, hydrophobic transition state for the reaction.« less

Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I

PubMed Central

Shi, Yuqian; Hellinga, Homme W.; Beese, Lorena S.

2017-01-01

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5′-nuclease superfamily. Its dominant, processive 5′–3′ exonuclease and secondary 5′-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5′-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5′ flaps. Their distinctive 5′ ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5′ flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity. PMID:28533382

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

PubMed

Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

2011-01-21

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Amyloidogenesis abolished by proline substitutions but enhanced by lipid binding.

PubMed

Jiang, Ping; Xu, Weixin; Mu, Yuguang

2009-04-01

The influence of lipid molecules on the aggregation of a highly amyloidogenic segment of human islet amyloid polypeptide, hIAPP20-29, and the corresponding sequence from rat has been studied by all-atom replica exchange molecular dynamics (REMD) simulations with explicit solvent model. hIAPP20-29 fragments aggregate into partially ordered beta-sheet oligomers and then undergo large conformational reorganization and convert into parallel/antiparallel beta-sheet oligomers in mixed in-register and out-of-register patterns. The hydrophobic interaction between lipid tails and residues at positions 23-25 is found to stabilize the ordered beta-sheet structure, indicating a catalysis role of lipid molecules in hIAPP20-29 self-assembly. The rat IAPP variants with three proline residues maintain unstructured micelle-like oligomers, which is consistent with non-amyloidogenic behavior observed in experimental studies. Our study provides the atomic resolution descriptions of the catalytic function of lipid molecules on the aggregation of IAPP peptides.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cavalier, Michael C.; Yim, Young-Sun; Asamizu, Shumpei

The pseudo-glycosyltransferase VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the stereochemical configuration of the substrates to form validoxylamine A 7'-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7'-phosphate, and in complex with GDP and trehalose. The structure of VldE with the catalytic site in both an “open” and “closed” conformation is also described. With thesemore » structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the stereochemical configuration of the donated cyclitol.« less

Characterizing substrate–surface interactions on alumina-supported metal catalysts by dynamic nuclear polarization-enhanced double-resonance NMR spectroscopy [Characterizing substrate-surface interactions on alumina supported metal catalysts by DNP-enhanced double-resonance NMR spectroscopy

DOE PAGES

Perras, Frederic A.; Padmos, J. Daniel; Johnson, Robert L.; ...

2017-01-23

The characterization of nanometer-scale interactions between carbon-containing substrates and alumina surfaces is of paramount importance to industrial and academic catalysis applications, but it is also very challenging. Here, we demonstrate that dynamic nuclear polarization surface-enhanced NMR spectroscopy (DNP SENS) allows the unambiguous description of the coordination geometries and conformations of the substrates at the alumina surface through high-resolution measurements of 13C– 27Al distances. We apply this new technique to elucidate the molecular-level geometry of 13C-enriched methionine and natural abundance poly(vinyl alcohol) adsorbed on γ-Al 2O 3-supported Pd catalysts, and we support these results with element-specific X-ray absorption near-edge measurements. Furthermore,more » this work clearly demonstrates a surprising bimodal coordination of methionine at the Pd–Al 2O 3 interface.« less

Characterizing substrate–surface interactions on alumina-supported metal catalysts by dynamic nuclear polarization-enhanced double-resonance NMR spectroscopy [Characterizing substrate-surface interactions on alumina supported metal catalysts by DNP-enhanced double-resonance NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perras, Frederic A.; Padmos, J. Daniel; Johnson, Robert L.

The characterization of nanometer-scale interactions between carbon-containing substrates and alumina surfaces is of paramount importance to industrial and academic catalysis applications, but it is also very challenging. Here, we demonstrate that dynamic nuclear polarization surface-enhanced NMR spectroscopy (DNP SENS) allows the unambiguous description of the coordination geometries and conformations of the substrates at the alumina surface through high-resolution measurements of 13C– 27Al distances. We apply this new technique to elucidate the molecular-level geometry of 13C-enriched methionine and natural abundance poly(vinyl alcohol) adsorbed on γ-Al 2O 3-supported Pd catalysts, and we support these results with element-specific X-ray absorption near-edge measurements. Furthermore,more » this work clearly demonstrates a surprising bimodal coordination of methionine at the Pd–Al 2O 3 interface.« less

Template-constrained macrocyclic peptides prepared from native, unprotected precursors

PubMed Central

Lawson, Kenneth V.; Rose, Tristan E.; Harran, Patrick G.

2013-01-01

Peptide–protein interactions are important mediators of cellular-signaling events. Consensus binding motifs (also known as short linear motifs) within these contacts underpin molecular recognition, yet have poor pharmacological properties as discrete species. Here, we present methods to transform intact peptides into stable, templated macrocycles. Two simple steps install the template. The key reaction is a palladium-catalyzed macrocyclization. The catalysis has broad scope and efficiently forms large rings by engaging native peptide functionality including phenols, imidazoles, amines, and carboxylic acids without the necessity of protecting groups. The tunable reactivity of the template gives the process special utility. Defined changes in reaction conditions markedly alter chemoselectivity. In all cases examined, cyclization occurs rapidly and in high yield at room temperature, regardless of peptide composition or chain length. We show that conformational restraints imparted by the template stabilize secondary structure and enhance proteolytic stability in vitro. Palladium-catalyzed internal cinnamylation is a strong complement to existing methods for peptide modification. PMID:24043790

Methyl Transfer by Substrate Signaling from a Knotted Protein Fold

PubMed Central

Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P.; Lahoud, Georges; Ito, Takuhiro; Taylor, Erika A.; Yokoyama, Shigeyuki; Sulkowska, Joanna I.; Hou, Ya-Ming

2017-01-01

Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis. PMID:27571175

Examination of key intermediates in the catalytic cycle of aspartate-beta-semialdehyde dehydrogenase from a gram-positive infectious bacteria.

PubMed

Faehnle, Christopher R; Le Coq, Johanne; Liu, Xuying; Viola, Ronald E

2006-10-13

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.

Integration process of biodiesel production from filamentous oleaginous microalgae Tribonema minus.

PubMed

Wang, Hui; Gao, Lili; Chen, Lin; Guo, Fajin; Liu, Tianzhong

2013-08-01

Biodiesel production from microalgae has been receiving considerable attention. Past studies mainly relied on tiny sized single-cell oleaginous microalgal species, the biodiesel based on filamentous oleaginous microalgae was rarely reported. Thus, integrated process of biodiesel production from filamentous oleaginous microalgal strain Tribonema minus was studied in this work. The filamentous microalgae was cultivated for 21 days in 40 L glass panel, microalgae cells was harvested by DAF without any flocculants after the lipid content was 50.23%. After that, total lipid was extracted by subcritical ethanol from wet algal paste and 44.55% of crude lipid was triacylglycerols. Two-step catalytic conversion of pre-esterification and transesterification was adopted to convert the crude algal oil to biodiesel. The conversion rate of triacylglycerols reached 96.52% under the methanol to oil molar ratio of 12:1 during catalysis with 2% potassium hydroxide at 65°C for 30 min. The biodiesel product from T. minus conformed to Chinese National Standards. Copyright © 2013 Elsevier Ltd. All rights reserved.

Amyloidogenesis Abolished by Proline Substitutions but Enhanced by Lipid Binding

PubMed Central

Jiang, Ping; Xu, Weixin; Mu, Yuguang

2009-01-01

The influence of lipid molecules on the aggregation of a highly amyloidogenic segment of human islet amyloid polypeptide, hIAPP20–29, and the corresponding sequence from rat has been studied by all-atom replica exchange molecular dynamics (REMD) simulations with explicit solvent model. hIAPP20–29 fragments aggregate into partially ordered β-sheet oligomers and then undergo large conformational reorganization and convert into parallel/antiparallel β-sheet oligomers in mixed in-register and out-of-register patterns. The hydrophobic interaction between lipid tails and residues at positions 23–25 is found to stabilize the ordered β-sheet structure, indicating a catalysis role of lipid molecules in hIAPP20–29 self-assembly. The rat IAPP variants with three proline residues maintain unstructured micelle-like oligomers, which is consistent with non-amyloidogenic behavior observed in experimental studies. Our study provides the atomic resolution descriptions of the catalytic function of lipid molecules on the aggregation of IAPP peptides. PMID:19360098

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huerta, Carlos; Borek, Dominika; Machius, Mischa

2009-12-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme flavin adenine dinucleotide (FAD) and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different sets of active-site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from the pathogenic yeast Candida glabrata. Four crystal structures of C. glabrata FMNAT in different complexed forms were determined at 1.20-1.95 A resolutions, capturing the enzyme active-site states prior to and after catalysis. These structures reveal a novelmore » flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNATs provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. Structure-based investigation of the kinetic properties of FMNAT should offer insights into the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.« less

Structure and mechanism of human DNA polymerase [eta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji

2010-11-03

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assistmore » translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.« less

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules.

PubMed

Windle, Claire L; Berry, Alan; Nelson, Adam

2017-04-01

The introduction of fluorine has been widely exploited to tune the biological functions of small molecules. Indeed, around 20% of leading drugs contain at least one fluorine atom. Yet, despite profound effects of fluorination on conformation, there is only a limited toolkit of reactions that enable stereoselective synthesis of fluorinated compounds. Aldolases are useful catalysts for the stereoselective synthesis of bioactive small molecules; however, despite fluoropyruvate being a viable nucleophile for some aldolases, the potential of aldolases to control the formation of fluorine-bearing stereocentres has largely been untapped. Very recently, it has been shown that aldolase-catalysed stereoselective carboncarbon bond formation with fluoropyruvate as nucleophile enable the synthesis of many α-fluoro β-hydroxy carboxyl derivatives. Furthermore, an understanding of the structural basis for the stereocontrol observed in these reactions is beginning to emerge. Here, we review the application of aldolase catalysis in the stereocontrolled synthesis of chiral fluorinated small molecules, and highlight likely areas for future developments. Copyright © 2017 Elsevier Ltd. All rights reserved.

From containers to catalysts: supramolecular catalysis within cucurbiturils.

PubMed

Pemberton, Barry C; Raghunathan, Ramya; Volla, Sabine; Sivaguru, Jayaraman

2012-09-24

Cucurbiturils are a family of molecular container compounds with superior molecular recognition properties. The use of cucurbiturils for supramolecular catalysis is highlighted in this concept. Both photochemical reactions as well as thermal transformations are reviewed with an eye towards tailoring substrates for supramolecular catalysis mediated by cucurbiturils. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study on the Application of the Combination of TMD Simulation and Umbrella Sampling in PMF Calculation for Molecular Conformational Transitions

PubMed Central

Wang, Qing; Xue, Tuo; Song, Chunnian; Wang, Yan; Chen, Guangju

2016-01-01

Free energy calculations of the potential of mean force (PMF) based on the combination of targeted molecular dynamics (TMD) simulations and umbrella samplings as a function of physical coordinates have been applied to explore the detailed pathways and the corresponding free energy profiles for the conformational transition processes of the butane molecule and the 35-residue villin headpiece subdomain (HP35). The accurate PMF profiles for describing the dihedral rotation of butane under both coordinates of dihedral rotation and root mean square deviation (RMSD) variation were obtained based on the different umbrella samplings from the same TMD simulations. The initial structures for the umbrella samplings can be conveniently selected from the TMD trajectories. For the application of this computational method in the unfolding process of the HP35 protein, the PMF calculation along with the coordinate of the radius of gyration (Rg) presents the gradual increase of free energies by about 1 kcal/mol with the energy fluctuations. The feature of conformational transition for the unfolding process of the HP35 protein shows that the spherical structure extends and the middle α-helix unfolds firstly, followed by the unfolding of other α-helices. The computational method for the PMF calculations based on the combination of TMD simulations and umbrella samplings provided a valuable strategy in investigating detailed conformational transition pathways for other allosteric processes. PMID:27171075

The topomer-sampling model of protein folding

PubMed Central

Debe, Derek A.; Carlson, Matt J.; Goddard, William A.

1999-01-01

Clearly, a protein cannot sample all of its conformations (e.g., ≈3100 ≈ 1048 for a 100 residue protein) on an in vivo folding timescale (<1 s). To investigate how the conformational dynamics of a protein can accommodate subsecond folding time scales, we introduce the concept of the native topomer, which is the set of all structures similar to the native structure (obtainable from the native structure through local backbone coordinate transformations that do not disrupt the covalent bonding of the peptide backbone). We have developed a computational procedure for estimating the number of distinct topomers required to span all conformations (compact and semicompact) for a polypeptide of a given length. For 100 residues, we find ≈3 × 107 distinct topomers. Based on the distance calculated between different topomers, we estimate that a 100-residue polypeptide diffusively samples one topomer every ≈3 ns. Hence, a 100-residue protein can find its native topomer by random sampling in just ≈100 ms. These results suggest that subsecond folding of modest-sized, single-domain proteins can be accomplished by a two-stage process of (i) topomer diffusion: random, diffusive sampling of the 3 × 107 distinct topomers to find the native topomer (≈0.1 s), followed by (ii) intratopomer ordering: nonrandom, local conformational rearrangements within the native topomer to settle into the precise native state. PMID:10077555

Color deviation controlling of phosphor conformal coating by advanced spray painting technology for white LEDs.

PubMed

Yang, Liang; Wang, Simin; Lv, Zhicheng; Liu, Sheng

2013-04-01

An advanced phosphor conformal coating technology is proposed, good correlated color temperature (CCT) and chromaticity uniformity samples are fabricated through phosphor spray painting technology. Spray painting technology is also suitable for phosphor conformal coating of whole LED wafers. The samples of different CCTs are obtained through controlling the phosphor film thickness in the range of 6-80 μm; CCT variation of samples can be controlled in the range of ±200 K. The experimental Δuv reveals that the spray painting method can obtain a much smaller CCT variation (Δuv of 1.36e(-3)) than the conventional dispensing method (Δuv of 11.86e(-3)) when the light is emitted at angles from -90° to +90°, and chromaticity area uniformity is also improved significantly.

Anharmonic Vibrational Analyses of Pentapeptide Conformations Explored with Enhanced Sampling Simulations.

PubMed

Otaki, Hiroki; Yagi, Kiyoshi; Ishiuchi, Shun-Ichi; Fujii, Masaaki; Sugita, Yuji

2016-10-06

An accurate theoretical prediction of the vibrational spectrum of polypeptides remains to be a challenge due to (1) their conformational flexibility and (2) non-negligible anharmonic effects. The former makes the search for conformers that contribute to the spectrum difficult, and the latter requires an expensive, quantum mechanical calculation for both electrons and vibrations. Here, we propose a new theoretical approach, which implements an enhanced conformational sampling by the replica-exchange molecular dynamics method, a structural clustering to identify distinct conformations, and a vibrational structure calculation by the second-order vibrational quasi-degenerate perturbation theory (VQDPT2). A systematic mode-selection scheme is developed to reduce the cost of VQDPT2 and the generation of a potential energy surface by the electronic structure calculation. The proposed method is applied to a pentapeptide, SIVSF-NH 2 , for which the infrared spectrum has recently been measured in the gas phase with high resolution in the OH and NH stretching region. The theoretical spectrum of the lowest energy conformer is obtained with a mean absolute deviation of 11.2 cm -1 from the experimental spectrum. Furthermore, the NH stretching frequencies of the five lowest energy conformers are found to be consistent with the literature values measured for small peptides with a similar secondary structure. Therefore, the proposed method is a promising way to analyze the vibrational spectrum of polypeptides.

LDRD 2013 Annual Report: Laboratory Directed Research and Development Program Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bookless, W.

This LDRD project establishes a research program led by Jingguang Chen, who has started a new position as a Joint Appointee between BNL and Columbia University as of FY2013. Under this project, Dr. Chen will establish a new program in catalysis science at BNL and Columbia University. The LDRD program will provide initial research funding to start research at both BNL and Columbia. At BNL, Dr. Chen will initiate laboratory research, including hiring research staff, and will collaborate with the existing BNL catalysis and electrocatalysis research groups. At Columbia, a subcontract to Dr. Chen will provide startup funding for hismore » laboratory research, including initial graduate student costs. The research efforts will be linked under a common Catalysis Program in Sustainable Fuels. The overall impact of this project will be to strengthen the BNL catalysis science program through new linked research thrusts and the addition of an internationally distinguished catalysis scientist.« less

Bioinspired construction of multi-enzyme catalytic systems.

PubMed

Shi, Jiafu; Wu, Yizhou; Zhang, Shaohua; Tian, Yu; Yang, Dong; Jiang, Zhongyi

2018-06-18

Enzyme catalysis, as a green, efficient process, displays exceptional functionality, adaptivity and sustainability. Multi-enzyme catalysis, which can accomplish the tandem synthesis of valuable materials/chemicals from renewable feedstocks, establishes a bridge between single-enzyme catalysis and whole-cell catalysis. Multi-enzyme catalysis occupies a unique and indispensable position in the realm of biological reactions for energy and environmental applications. Two complementary strategies, i.e., compartmentalization and substrate channeling, have been evolved by living organisms for implementing the complex in vivo multi-enzyme reactions (MERs), which have been applied to construct multi-enzyme catalytic systems (MECSs) with superior catalytic activity and stabilities in practical biocatalysis. This tutorial review aims to present the recent advances and future prospects in this burgeoning research area, stressing the features and applications of the two strategies for constructing MECSs and implementing in vitro MERs. The concluding remarks are presented with a perspective on the construction of MECSs through rational combination of compartmentalization and substrate channeling.

Toward benchmarking in catalysis science: Best practices, challenges, and opportunities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bligaard, Thomas; Bullock, R. Morris; Campbell, Charles T.

Benchmarking is a community-based and (preferably) community-driven activity involving consensus-based decisions on how to make reproducible, fair, and relevant assessments. In catalysis science, important catalyst performance metrics include activity, selectivity, and the deactivation profile, which enable comparisons between new and standard catalysts. Benchmarking also requires careful documentation, archiving, and sharing of methods and measurements, to ensure that the full value of research data can be realized. Beyond these goals, benchmarking presents unique opportunities to advance and accelerate understanding of complex reaction systems by combining and comparing experimental information from multiple, in situ and operando techniques with theoretical insights derived frommore » calculations characterizing model systems. This Perspective describes the origins and uses of benchmarking and its applications in computational catalysis, heterogeneous catalysis, molecular catalysis, and electrocatalysis. As a result, it also discusses opportunities and challenges for future developments in these fields.« less

Toward benchmarking in catalysis science: Best practices, challenges, and opportunities

DOE PAGES

Bligaard, Thomas; Bullock, R. Morris; Campbell, Charles T.; ...

2016-03-07

Benchmarking is a community-based and (preferably) community-driven activity involving consensus-based decisions on how to make reproducible, fair, and relevant assessments. In catalysis science, important catalyst performance metrics include activity, selectivity, and the deactivation profile, which enable comparisons between new and standard catalysts. Benchmarking also requires careful documentation, archiving, and sharing of methods and measurements, to ensure that the full value of research data can be realized. Beyond these goals, benchmarking presents unique opportunities to advance and accelerate understanding of complex reaction systems by combining and comparing experimental information from multiple, in situ and operando techniques with theoretical insights derived frommore » calculations characterizing model systems. This Perspective describes the origins and uses of benchmarking and its applications in computational catalysis, heterogeneous catalysis, molecular catalysis, and electrocatalysis. As a result, it also discusses opportunities and challenges for future developments in these fields.« less

Gender conformity, self-objectification, and body image for sorority and nonsorority women: A closer look.

PubMed

Adams, David Francis; Behrens, Erica; Gann, Lianne; Schoen, Eva

2017-01-01

Sororities have been identified as placing young women at risk for body image concerns due to a focus on traditional gender role norms and objectification of women. This study assessed the relationship between conformity to feminine gender role norms, self-objectification, and body image surveillance among undergraduate women. In a random sample of undergraduates, the authors examined data from sorority and nonsorority women. In a random sample of undergraduate women, the authors assessed the impact of traditional feminine gender role norms on self-objectification, body image, and feedback regarding physical appearance for sorority and nonsorority undergraduate women. Three linear regressions were conducted, and only conformity to feminine gender role norms contributed significantly in each regression model. Regardless of sorority membership, conformity to feminine gender role norms was found to significantly contribute to increased body consciousness, negative body image, and feedback on physical appearance.

Influence of substituents on the solution conformation of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9.

PubMed

Gianni, R; Cescutti, P; Bosco, M; Fett, W F; Rizzo, R

1999-12-01

The influence of pyruvate ketals and acetyl groups on the conformational behaviour of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9 has been investigated experimentally through studies of intrinsic viscosity and circular dichroism experiments. A conformational variation was detected as a function of the ionic strength. Measurements carried out on the native polymer, as well as on both de-pyruvated and de-acetylated samples, suggested a critical role for the acetyl group on the solution conformation of the polysaccharide. Molecular mechanics calculations indicated the possibility of intramolecular hydrogen bonding between acetyl substituents on the mannose and the C(2)OH group of the preceding saccharidic unit. NMR linewidth measurements, carried out as a function of temperature, on the low molecular weight de-pyruvated sample indicated different polymeric backbone dynamics in aqueous solutions with respect to that observed in 0.3 M NaCl solutions.

Photoacoustic spectroscopy of condensed matter

NASA Technical Reports Server (NTRS)

Somoano, R. B.

1978-01-01

Photoacoustic spectroscopy is a new analytical tool that provides a simple nondestructive technique for obtaining information about the electronic absorption spectrum of samples such as powders, semisolids, gels, and liquids. It can also be applied to samples which cannot be examined by conventional optical methods. Numerous applications of this technique in the field of inorganic and organic semiconductors, biology, and catalysis have been described. Among the advantages of photoacoustic spectroscopy, the signal is almost insensitive to light scattering by the sample and information can be obtained about nonradiative deactivation processes. Signal saturation, which can modify the intensity of individual absorption bands in special cases, is a drawback of the method.

Backbone conformations and side chain flexibility of two somatostatin mimics investigated by molecular dynamics simulations.

PubMed

Interlandi, Gianluca

2009-05-15

Molecular dynamics simulations with two designed somatostatin mimics, SOM230 and SMS 201-995, were performed in explicit water for a total aggregated time of 208 ns. Analysis of the runs with SOM230 revealed the presence of two clusters of conformations. Strikingly, the two sampled conformers correspond to the two main X-ray structures in the asymmetric unit of SMS 201-995. Structural comparison between the residues of SOM230 and SMS 201-995 provides an explanation for the high binding affinity of SOM230 to four of five somatostatin receptors. Similarly, cluster analysis of the simulations with SMS 201-995 shows that the backbone of the peptide interconverts between its two main crystallographic conformers. The conformations of SMS 201-995 sampled in the two clusters violated two different sets of NOE distance constraints in agreement with a previous NMR study. Differences in side chain fluctuations between SOM230 and SMS 201-995 observed in the simulations may contribute to the relatively higher binding affinity of SOM230 to most somatostatin receptors.

Tandem catalysis: a new approach to polymers.

PubMed

Robert, Carine; Thomas, Christophe M

2013-12-21

The creation of polymers by tandem catalysis represents an exciting frontier in materials science. Tandem catalysis is one of the strategies used by Nature for building macromolecules. Living organisms generally synthesize macromolecules by in vivo enzyme-catalyzed chain growth polymerization reactions using activated monomers that have been formed within cells during complex metabolic processes. However, these biological processes rely on highly complex biocatalysts, thus limiting their industrial applications. In order to obtain polymers by tandem catalysis, homogeneous and enzyme catalysts have played a leading role in the last two decades. In the following feature article, we will describe selected published efforts to achieve these research goals.

Catalysis and biocatalysis program

NASA Technical Reports Server (NTRS)

1991-01-01

The annual report presents the fiscal year (FY) 1990 research activities and accomplishments for the Catalysis and Biocatalysis Program of the Advanced Industrial Concepts Division (AICD), Office of Industrial Technologies of the Department of Energy (DOE). The mission of the AICD is to create a balanced program of high risk, long term, directed interdisciplinary research and development that will improve energy efficiency and enhance fuel flexibility in the industrial sector. The Catalysis and Biocatalysis Program's technical activities were organized into five work elements: the Molecular Modeling and Catalysis by Design element; the Applied Microbiology and Genetics element; the Bioprocess Engineering element; the Separations and Novel Chemical Processes element; and the Process Design and Analysis element.

Multielectron, multisubstrate molecular catalysis of electrochemical reactions: Formal kinetic analysis in the total catalysis regime.

PubMed

Costentin, Cyrille; Nocera, Daniel G; Brodsky, Casey N

2017-10-24

Cyclic voltammetry responses are derived for two-electron, two-step homogeneous electrocatalytic reactions in the total catalysis regime. The models developed provide a framework for extracting kinetic information from cyclic voltammograms (CVs) obtained in conditions under which the substrate or cosubstrate is consumed in a multielectron redox process, as is particularly prevalent for very active catalysts that promote energy conversion reactions. Such determination of rate constants in the total catalysis regime is a prerequisite for the rational benchmarking of molecular electrocatalysts that promote multielectron conversions of small-molecule reactants. The present analysis is illustrated with experimental systems encompassing various limiting behaviors.

First kinetic discrimination between carbon and oxygen reactivity of enols.

PubMed

García-Río, Luis; Mejuto, Juan C; Parajó, Mercedes; Pérez-Lorenzo, Moisés

2008-11-07

Nitrosation of enols shows a well-differentiated behavior depending on whether the reaction proceeds through the carbon (nucleophilic catalysis is observed) or the oxygen atom (general acid-base catalysis is observed). This is due to the different operating mechanisms for C- and O-nitrosation. Nitrosation of acetylacetone (AcAc) shows a simultaneous nucleophilic and acid-base catalysis. This simultaneous catalysis constitutes the first kinetic evidence of two independent reactions on the carbon and oxygen atom of an enol. The following kinetic study allows us to determine the rate constants for both reaction pathways. A similar reactivity of the nucleophilic centers with the nitrosonium ion is observed.

Iminium and enamine catalysis in enantioselective photochemical reactions.

PubMed

Zou, You-Quan; Hörmann, Fabian M; Bach, Thorsten

2018-01-22

Although enantioselective catalysis under thermal conditions has been well established over the last few decades, the enantioselective catalysis of photochemical reactions is still a challenging task resulting from the complex enantiotopic face differentiation in the photoexcited state. Recently, remarkable achievements have been reported by a synergistic combination of organocatalysis and photocatalysis, which have led to the expedient construction of a diverse range of enantioenriched molecules which are generally not easily accessible under thermal conditions. In this tutorial review, we summarize and highlight the most significant advances in iminium and enamine catalysis of enantioselective photochemical reactions, with an emphasis on catalytic modes and reaction types.

Iminium and enamine catalysis in enantioselective photochemical reactions

PubMed Central

Hörmann, Fabian M.

2018-01-01

Although enantioselective catalysis under thermal conditions has been well established over the last few decades, the enantioselective catalysis of photochemical reactions is still a challenging task resulting from the complex enantiotopic face differentiation in the photoexcited state. Recently, remarkable achievements have been reported by a synergistic combination of organocatalysis and photocatalysis, which have led to the expedient construction of a diverse range of enantioenriched molecules which are generally not easily accessible under thermal conditions. In this tutorial review, we summarize and highlight the most significant advances in iminium and enamine catalysis of enantioselective photochemical reactions, with an emphasis on catalytic modes and reaction types. PMID:29155908

A kinetic study on the catalysis of KCl, K2SO4, and K2CO3 during oxy-biomass combustion.

PubMed

Deng, Shuanghui; Wang, Xuebin; Zhang, Jiaye; Liu, Zihan; Mikulčić, Hrvoje; Vujanović, Milan; Tan, Houzhang; Duić, Neven

2018-07-15

Biomass combustion under the oxy-fuel conditions (Oxy-biomass combustion) is one of the approaches achieving negative CO 2 emissions. KCl, K 2 CO 3 and K 2 SO 4 , as the major potassium species in biomass ash, can catalytically affect biomass combustion. In this paper, the catalysis of the representative potassium salts on oxy-biomass combustion was studied using a thermogravimetric analyzer (TGA). Effects of potassium salt types (KCl, K 2 CO 3 and K 2 SO 4 ), loading concentrations (0, 1, 3, 5, 8 wt%), replacing N 2 by CO 2 , and O 2 concentrations (5, 20, 30 vol%) on the catalysis degree were discussed. The comparison between TG-DTG curves of biomass combustion before and after water washing in both the 20%O 2 /80%N 2 and 20%O 2 /80%CO 2 atmospheres indicates that the water-soluble minerals in biomass play a role in promoting the devolatilization and accelerating the char-oxidation; and the replacement of N 2 by CO 2 inhibits the devolatilization and char-oxidation processes during oxy-biomass combustion. In the devolatilization stage, the catalysis degree of potassium monotonously increases with the increase of potassium salt loaded concentration. The catalysis degree order of the studied potassium salts is K 2 CO 3  > KCl > K 2 SO 4 . In the char-oxidation stage, with the increase of loading concentration the three kinds of potassium salts present inconsistent change tendencies of the catalysis degree. In the studied loading concentrations from 0 to 8 wt%, there is an optimal loading concentration for KCl and K 2 CO 3 , at 3 and 5 wt%, respectively; while for K 2 SO 4 , the catalysis degree on char-oxidation monotonically increases with the loading potassium concentration. For most studied conditions, regardless of the potassium salt types or the loading concentrations or the combustion stages, the catalysis degree in the O 2 /CO 2 atmosphere is stronger than that in the O 2 /N 2 atmosphere. The catalysis degree is also affected by the O 2 concentrations, and the lowest catalysis degree is generally around 20 vol% O 2 concentration. The kinetic parameters under the different studied conditions are finally obtained. Copyright © 2018 Elsevier Ltd. All rights reserved.

Exploring Flexibility of Progesterone Receptor Ligand Binding Domain Using Molecular Dynamics

PubMed Central

Zheng, Liangzhen; Mu, Yuguang

2016-01-01

Progesterone receptor (PR), a member of nuclear receptor (NR) superfamily, plays a vital role for female reproductive tissue development, differentiation and maintenance. PR ligand, such as progesterone, induces conformation changes in PR ligand binding domain (LBD), thus mediates subsequent gene regulation cascades. PR LBD may adopt different conformations upon an agonist or an antagonist binding. These different conformations would trigger distinct transcription events. Therefore, the dynamics of PR LBD would be of general interest to biologists for a deep understanding of its structure-function relationship. However, no apo-form (non-ligand bound) of PR LBD model has been proposed either by experiments or computational methods so far. In this study, we explored the structural dynamics of PR LBD using molecular dynamics simulations and advanced sampling tools in both ligand-bound and the apo-forms. Resolved by the simulation study, helix 11, helix 12 and loop 895–908 (the loop between these two helices) are quite flexible in antagonistic conformation. Several residues, such as Arg899 and Glu723, could form salt-bridging interaction between helix 11 and helix 3, and are important for the PR LBD dynamics. And we also propose that helix 12 in apo-form PR LBD, not like other NR LBDs, such as human estrogen receptor α (ERα) LBD, may not adopt a totally extended conformation. With the aid of umbrella sampling and metadynamics simulations, several stable conformations of apo-form PR LBD have been sampled, which may work as critical structural models for further large scale virtual screening study to discover novel PR ligands for therapeutic application. PMID:27824891

Computational Analysis of a Zn-Bound Tris(imidazolyl) Calix[6]arene Aqua Complex: Toward Incorporating Second-Coordination Sphere Effects into Carbonic Anhydrase Biomimetics.

PubMed

Koziol, Lucas; Essiz, Sebnem G; Wong, Sergio E; Lau, Edmond Y; Valdez, Carlos A; Satcher, Joe H; Aines, Roger D; Lightstone, Felice C

2013-03-12

Molecular dynamics simulations and quantum-mechanical calculations were performed to characterize a supramolecular tris(imidazolyl) calix[6]arene Zn(2+) aqua complex, as a biomimetic model for the catalyzed hydration of carbon dioxide to bicarbonate, H2O + CO2 → H(+) + HCO3(-). On the basis of potential-of-mean-force (PMF) calculations, stable conformations had distorted 3-fold symmetry and supported either one or zero encapsulated water molecules. The conformation with an encapsulated water molecule is calculated to be lower in free energy than the conformation with an empty cavity (ΔG = 1.2 kcal/mol) and is the calculated free-energy minimum in solution. CO2 molecule partitioning into the cavity is shown to be very facile, proceeding with a barrier of 1.6 kcal/mol from a weak encounter complex which stabilizes the species by about 1.0 kcal/mol. The stabilization energy of CO2 is calculated to be larger than that of H2O (ΔΔG = 1.4 kcal/mol), suggesting that the complex will preferentially encapsulate CO2 in solution. In contrast, the PMF for a bicarbonate anion entering the cavity is calculated to be repulsive in all nonbonding regions of the cavity, due to the diameter of the calix[6]arene walls. Geometry optimization of the Zn-bound hydroxide complex with an encapsulated CO2 molecule showed that multiple noncovalent interactions direct the reactants into optimal position for nucleophilic addition to occur. The calixarene complex is a structural mimic of the hydrophilic/hydrophobic divide in the enzyme, providing a functional effect for CO2 addition in the catalytic cycle. The results show that Zn-binding calix[6]arene scaffolds can be potential synthetic biomimetics for CO2 hydration catalysis, both in terms of preferentially encapsulating CO2 from solution and by spatially fixing the reactive species inside the cavity.

Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation

PubMed Central

Okuno, Daichi; Fujisawa, Ryo; Iino, Ryota; Hirono-Hara, Yoko; Imamura, Hiromi; Noji, Hiroyuki

2008-01-01

F1-ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α3β3 ring. The crystal structures of F1, which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F1, it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F1, the corresponding residues in the ADP-bound β (βDP) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F1, α3β2β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F1 paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that βDP is the catalytically active form. The former point was again confirmed in experiments where F1 rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F1. PMID:19075235

On the Use of Enveloping Distribution Sampling (EDS) to Compute Free Enthalpy Differences between Different Conformational States of Molecules: Application to 310-, α-, and π-Helices.

PubMed

Lin, Zhixiong; Liu, Haiyan; Riniker, Sereina; van Gunsteren, Wilfred F

2011-12-13

Enveloping distribution sampling (EDS) is a powerful method to compute relative free energies from simulation. So far, the EDS method has only been applied to alchemical free energy differences, i.e., between different Hamiltonians defining different systems, and not yet to obtain free energy differences between different conformations or conformational states of a system. In this article, we extend the EDS formalism such that it can be applied to compute free energy differences of different conformations and apply it to compute the relative free enthalpy ΔG of 310-, α-, and π-helices of an alanine deca-peptide in explicit water solvent. The resulting ΔG values are compared to those obtained by standard thermodynamic integration (TI) and from so-called end-state simulations. A TI simulation requires the definition of a λ-dependent pathway which in the present case is based on hydrogen bonds of the different helical conformations. The values of ⟨(∂VTI)/(∂λ)⟩λ show a sharp change for a particular range of λ values, which is indicative of an energy barrier along the pathway, which lowers the accuracy of the resulting ΔG value. In contrast, in a two-state EDS simulation, an unphysical reference-state Hamiltonian which connects the parts of conformational space that are relevant to the different end states is constructed automatically; that is, no pathway needs to be defined. In the simulation using this reference state, both helices were sampled, and many transitions between them occurred, thus ensuring the accuracy of the resulting free enthalpy difference. According to the EDS simulations, the free enthalpy differences of the π-helix and the 310-helix versus the α-helix are 5 kJ mol(-1) and 47 kJ mol(-1), respectively, for an alanine deca-peptide in explicit SPC water solvent using the GROMOS 53A6 force field. The EDS method, which is a particular form of umbrella sampling, is thus applicable to compute free energy differences between conformational states as well as between systems and has definite advantages over the traditional TI and umbrella sampling methods to compute relative free energies.

Elucidating the ensemble of functionally-relevant transitions in protein systems with a robotics-inspired method

PubMed Central

2013-01-01

Background Many proteins tune their biological function by transitioning between different functional states, effectively acting as dynamic molecular machines. Detailed structural characterization of transition trajectories is central to understanding the relationship between protein dynamics and function. Computational approaches that build on the Molecular Dynamics framework are in principle able to model transition trajectories at great detail but also at considerable computational cost. Methods that delay consideration of dynamics and focus instead on elucidating energetically-credible conformational paths connecting two functionally-relevant structures provide a complementary approach. Effective sampling-based path planning methods originating in robotics have been recently proposed to produce conformational paths. These methods largely model short peptides or address large proteins by simplifying conformational space. Methods We propose a robotics-inspired method that connects two given structures of a protein by sampling conformational paths. The method focuses on small- to medium-size proteins, efficiently modeling structural deformations through the use of the molecular fragment replacement technique. In particular, the method grows a tree in conformational space rooted at the start structure, steering the tree to a goal region defined around the goal structure. We investigate various bias schemes over a progress coordinate for balance between coverage of conformational space and progress towards the goal. A geometric projection layer promotes path diversity. A reactive temperature scheme allows sampling of rare paths that cross energy barriers. Results and conclusions Experiments are conducted on small- to medium-size proteins of length up to 214 amino acids and with multiple known functionally-relevant states, some of which are more than 13Å apart of each-other. Analysis reveals that the method effectively obtains conformational paths connecting structural states that are significantly different. A detailed analysis on the depth and breadth of the tree suggests that a soft global bias over the progress coordinate enhances sampling and results in higher path diversity. The explicit geometric projection layer that biases the exploration away from over-sampled regions further increases coverage, often improving proximity to the goal by forcing the exploration to find new paths. The reactive temperature scheme is shown effective in increasing path diversity, particularly in difficult structural transitions with known high-energy barriers. PMID:24565158

Sampling Molecular Conformers in Solution with Quantum Mechanical Accuracy at a Nearly Molecular-Mechanics Cost.

PubMed

Rosa, Marta; Micciarelli, Marco; Laio, Alessandro; Baroni, Stefano

2016-09-13

We introduce a method to evaluate the relative populations of different conformers of molecular species in solution, aiming at quantum mechanical accuracy, while keeping the computational cost at a nearly molecular-mechanics level. This goal is achieved by combining long classical molecular-dynamics simulations to sample the free-energy landscape of the system, advanced clustering techniques to identify the most relevant conformers, and thermodynamic perturbation theory to correct the resulting populations, using quantum-mechanical energies from density functional theory. A quantitative criterion for assessing the accuracy thus achieved is proposed. The resulting methodology is demonstrated in the specific case of cyanin (cyanidin-3-glucoside) in water solution.

Regulated bioanalysis of conformers - A case study with ASP2151 in dog plasma and urine.

PubMed

Ohtsu, Yoshiaki; Otsuka, Shohei; Nakamura, Takeshi; Noguchi, Kiyoshi

2015-08-01

We developed and validated bioanalytical methods for a potent helicase-primase inhibitor ASP2151 that has two conformers. These conformers elute as unseparated broad peaks under ordinary high-performance liquid chromatographic conditions, indicating discernable differences in hydrophobicity. We observed that column temperature and mobile phase pH have no effect on these peaks and that conformers form a single symmetrical peak when tetrahydrofuran is added to the mobile phase. In addition, we needed to develop semi-automated methods where inter-conversion of the conformers is unlikely to cause sample-to-sample extraction variability. Briefly, following the addition of deuterium-labeled ASP2151 as an internal standard (IS), dog plasma samples or acetonitrile-added urine samples were filtrated. The filtrates were then injected into a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and trapped onto an extraction column. Extracts were back-flushed onto an analytical C18 column (4.6×50mm, 3μm) with a mobile phase consisting of methanol, tetrahydrofuran, and 20mmol/L ammonium acetate (45:5:50, v/v/v). The eluent was monitored in the negative atmospheric pressure chemical ionization mode. The calibration curve was linear over a range of 5-1000ng/mL for plasma and 0.5-100μg/mL for urine. Validation data met the acceptance criteria in accordance with regulatory guidance and demonstrated that these methods were selective, accurate, and reproducible. In addition, the present methods were successfully applied to a pharmacokinetic study in dogs. Copyright © 2015 Elsevier B.V. All rights reserved.

PubChem3D: conformer ensemble accuracy

PubMed Central

2013-01-01

Background PubChem is a free and publicly available resource containing substance descriptions and their associated biological activity information. PubChem3D is an extension to PubChem containing computationally-derived three-dimensional (3-D) structures of small molecules. All the tools and services that are a part of PubChem3D rely upon the quality of the 3-D conformer models. Construction of the conformer models currently available in PubChem3D involves a clustering stage to sample the conformational space spanned by the molecule. While this stage allows one to downsize the conformer models to more manageable size, it may result in a loss of the ability to reproduce experimentally determined “bioactive” conformations, for example, found for PDB ligands. This study examines the extent of this accuracy loss and considers its effect on the 3-D similarity analysis of molecules. Results The conformer models consisting of up to 100,000 conformers per compound were generated for 47,123 small molecules whose structures were experimentally determined, and the conformers in each conformer model were clustered to reduce the size of the conformer model to a maximum of 500 conformers per molecule. The accuracy of the conformer models before and after clustering was evaluated using five different measures: root-mean-square distance (RMSD), shape-optimized shape-Tanimoto (STST-opt) and combo-Tanimoto (ComboTST-opt), and color-optimized color-Tanimoto (CTCT-opt) and combo-Tanimoto (ComboTCT-opt). On average, the effect of clustering decreased the conformer model accuracy, increasing the conformer ensemble’s RMSD to the bioactive conformer (by 0.18 ± 0.12 Å), and decreasing the STST-opt, ComboTST-opt, CTCT-opt, and ComboTCT-opt scores (by 0.04 ± 0.03, 0.16 ± 0.09, 0.09 ± 0.05, and 0.15 ± 0.09, respectively). Conclusion This study shows the RMSD accuracy performance of the PubChem3D conformer models is operating as designed. In addition, the effect of PubChem3D sampling on 3-D similarity measures shows that there is a linear degradation of average accuracy with respect to molecular size and flexibility. Generally speaking, one can likely expect the worst-case minimum accuracy of 90% or more of the PubChem3D ensembles to be 0.75, 1.09, 0.43, and 1.13, in terms of STST-opt, ComboTST-opt, CTCT-opt, and ComboTCT-opt, respectively. This expected accuracy improves linearly as the molecule becomes smaller or less flexible. PMID:23289532

Toward canonical ensemble distribution from self-guided Langevin dynamics simulation

NASA Astrophysics Data System (ADS)

Wu, Xiongwu; Brooks, Bernard R.

2011-04-01

This work derives a quantitative description of the conformational distribution in self-guided Langevin dynamics (SGLD) simulations. SGLD simulations employ guiding forces calculated from local average momentums to enhance low-frequency motion. This enhancement in low-frequency motion dramatically accelerates conformational search efficiency, but also induces certain perturbations in conformational distribution. Through the local averaging, we separate properties of molecular systems into low-frequency and high-frequency portions. The guiding force effect on the conformational distribution is quantitatively described using these low-frequency and high-frequency properties. This quantitative relation provides a way to convert between a canonical ensemble and a self-guided ensemble. Using example systems, we demonstrated how to utilize the relation to obtain canonical ensemble properties and conformational distributions from SGLD simulations. This development makes SGLD not only an efficient approach for conformational searching, but also an accurate means for conformational sampling.

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains.

PubMed

Kniss, Andreas; Schuetz, Denise; Kazemi, Sina; Pluska, Lukas; Spindler, Philipp E; Rogov, Vladimir V; Husnjak, Koraljka; Dikic, Ivan; Güntert, Peter; Sommer, Thomas; Prisner, Thomas F; Dötsch, Volker

2018-02-06

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational free energy modeling of druglike molecules by metadynamics in the WHIM space.

PubMed

Spiwok, Vojtěch; Hlat-Glembová, Katarína; Tvaroška, Igor; Králová, Blanka

2012-03-26

Protein-ligand affinities can be significantly influenced not only by the interaction itself but also by conformational equilibrium of both binding partners, free ligand and free protein. Identification of important conformational families of a ligand and prediction of their thermodynamics is important for efficient ligand design. Here we report conformational free energy modeling of nine small-molecule drugs in explicitly modeled water by metadynamics with a bias potential applied in the space of weighted holistic invariant molecular (WHIM) descriptors. Application of metadynamics enhances conformational sampling compared to unbiased molecular dynamics simulation and allows to predict relative free energies of key conformations. Selected free energy minima and one example of transition state were tested by a series of unbiased molecular dynamics simulation. Comparison of free energy surfaces of free and target-bound Imatinib provides an estimate of free energy penalty of conformational change induced by its binding to the target. © 2012 American Chemical Society