Lift-off process with bi-layer photoresist patterns for conformal-coated superhydrophilic pulsed plasma chemical vapor deposition-SiOx on SiCx for lab-on-a-chip applications

NASA Astrophysics Data System (ADS)

Konishi, Satoshi; Nakagami, Chise; Kobayashi, Taizo; Tonomura, Wataru; Kaizuma, Yoshihiro

2015-04-01

In this work, a lift-off process with bi-layer photoresist patterns was applied to the formation of hydrophobic/hydrophilic micropatterns on practical polymer substrates used in healthcare diagnostic commercial products. The bi-layer photoresist patterns with undercut structures made it possible to peel the conformal-coated silicon oxide (SiOx) films from substrates. SiOx and silicon carbide (SiCx) layers were deposited by pulsed plasma chemical vapor deposition (PPCVD) method which can form roughened surfaces to enhance hydrophilicity of SiOx and hydrophobicity of SiCx. Microfluidic applications using hydrophobic/hydrophilic patterns were also demonstrated on low-cost substrates such as poly(ethylene terephthalate) (PET) and paper films.

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.

A tunable sub-100 nm silicon nanopore array with an AAO membrane mask: reducing unwanted surface etching by introducing a PMMA interlayer.

PubMed

Lim, Namsoo; Pak, Yusin; Kim, Jin Tae; Hwang, Youngkyu; Lee, Ryeri; Kumaresan, Yogeenth; Myoung, NoSoung; Ko, Heung Cho; Jung, Gun Young

2015-08-28

Highly ordered silicon (Si) nanopores with a tunable sub-100 nm diameter were fabricated by a CF4 plasma etching process using an anodic aluminum oxide (AAO) membrane as an etching mask. To enhance the conformal contact of the AAO membrane mask to the underlying Si substrate, poly(methyl methacrylate) (PMMA) was spin-coated on top of the Si substrate prior to the transfer of the AAO membrane. The AAO membrane mask was fabricated by two-step anodization and subsequent removal of the aluminum support and the barrier layer, which was then transferred to the PMMA-coated Si substrate. Contact printing was performed on the sample with a pressure of 50 psi and a temperature of 120 °C to make a conformal contact of the AAO membrane mask to the Si substrate. The CF4 plasma etching was conducted to transfer nanopores onto the Si substrate through the PMMA interlayer. The introduced PMMA interlayer prevented unwanted surface etching of the Si substrate by eliminating the etching ions and radicals bouncing at the gap between the mask and the substrate, resulting in a smooth Si nanopore array.

Substrate transport pathway inside outward open conformation of EmrD: a molecular dynamics simulation study.

PubMed

Xianwei, Tan; Diannan, Lu; Boxiong, Wang

2016-07-19

The EmrD transporter, which is a classical major facilitator superfamily (MFS) protein, can extrude a range of drug molecules out of E. coil. The drug molecules transport through the channel of MFS in an outward open state, an important issue in research about bacterial drug resistance, which however, is still unknown. In this paper, we construct a starting outward-open model of the EmrD transporter using a state transition method. The starting model is refined by a conventional molecular dynamics simulation. Locally enhanced sampling simulation (LES) is used to validate the outward-open model of EmrD. In the locally enhanced sampling simulation, ten substrates are placed along the channel of the outward-open EmrD, and these substrates are sampled in the outward-open center cavity. It is found that the translocation pathway of these substrates from the inside to the outside of the cell through the EmrD transporter is composed of two sub-pathways, one sub-pathway, including H2, H4, and H5, and another sub-pathway, including H8, H10, and H11. The results give us have a further insight to the ways of substrate translocation of an MFS protein. The model method is based on common features of an MFS protein, so this modeling method can be used to construct various MFS protein models which have a desired state with other conformations not known in the alternating-access mechanism.

Informational and Normative Influences in Conformity from a Neurocomputational Perspective.

PubMed

Toelch, Ulf; Dolan, Raymond J

2015-10-01

We consider two distinct influences that drive conformity behaviour. Whereas informational influences facilitate adaptive and accurate responses, normative influences bias decisions to enhance social acceptance. We explore these influences from a perspective of perceptual and value-based decision-making models and apply these models to classical works on conformity. We argue that an informational account predicts a surprising tendency to conform. Moreover, we detail how normative influences fit into this framework and interact with social influences. Finally, we explore potential neuronal substrates for informational and normative influences based on a consideration of the neurobiological literature, highlighting conceptual shortcomings particularly with regard to a failure to segregate informational and normative influences. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conformational Plasticity of an Enzyme during Catalysis: Intricate Coupling between Cyclophilin A Dynamics and Substrate Turnover

PubMed Central

McGowan, Lauren C.; Hamelberg, Donald

2013-01-01

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. PMID:23332074

Conformational plasticity of an enzyme during catalysis: intricate coupling between cyclophilin A dynamics and substrate turnover.

PubMed

McGowan, Lauren C; Hamelberg, Donald

2013-01-08

Enzyme catalysis is central to almost all biochemical processes, speeding up rates of reactions to biological relevant timescales. Enzymes make use of a large ensemble of conformations in recognizing their substrates and stabilizing the transition states, due to the inherent dynamical nature of biomolecules. The exact role of these diverse enzyme conformations and the interplay between enzyme conformational dynamics and catalysis is, according to the literature, not well understood. Here, we use molecular dynamics simulations to study human cyclophilin A (CypA), in order to understand the role of enzyme motions in the catalytic mechanism and recognition. Cyclophilin A is a tractable model system to study using classical simulation methods, because catalysis does not involve bond formation or breakage. We show that the conformational dynamics of active site residues of substrate-bound CypA is inherent in the substrate-free enzyme. CypA interacts with its substrate via conformational selection as the configurations of the substrate changes during catalysis. We also show that, in addition to tight intermolecular hydrophobic interactions between CypA and the substrate, an intricate enzyme-substrate intermolecular hydrogen-bonding network is extremely sensitive to the configuration of the substrate. These enzyme-substrate intermolecular interactions are loosely formed when the substrate is in the reactant and product states and become well formed and reluctant to break when the substrate is in the transition state. Our results clearly suggest coupling among enzyme-substrate intermolecular interactions, the dynamics of the enzyme, and the chemical step. This study provides further insights into the mechanism of peptidyl-prolyl cis/trans isomerases and the general interplay between enzyme conformational dynamics and catalysis. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

PubMed Central

Menon, Binuraj R K; Menon, Navya; Fisher, Karl; Rigby, Stephen E J; Leys, David; Scrutton, Nigel S

2015-01-01

How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis. PMID:25627283

Mechanistic insights into Pin1 peptidyl-prolyl cis-trans isomerization from umbrella sampling simulations.

PubMed

Di Martino, Giovanni Paolo; Masetti, Matteo; Cavalli, Andrea; Recanatini, Maurizio

2014-11-01

The peptidyl-proyl isomerase Pin1 plays a key role in the regulation of phospho(p)-Ser/Thr-Pro proteins, acting as a molecular timer of the cell cycle. After recognition of these motifs, Pin1 catalyzes the rapid cis-trans isomerization of proline amide bonds of substrates, contributing to maintain the equilibrium between the two conformations. Although a great interest has arisen on this enzyme, its catalytic mechanism has long been debated. Here, the cis-trans isomerization of a model peptide system was investigated by means of umbrella sampling simulations in the Pin1-bound and unbound states. We obtained free energy barriers consistent with experimental data, and identified several enzymatic features directly linked to the acceleration of the prolyl bond isomerization. In particular, an enhanced autocatalysis, the stabilization of perturbed ground state conformations, and the substrate binding in a procatalytic conformation were found as main contributions to explain the lowering of the isomerization free energy barrier. © 2014 Wiley Periodicals, Inc.

Tip-enhanced Raman scattering of DNA aptamers for Listeria monocytogenes.

PubMed

He, Siyu; Li, Hongyuan; Gomes, Carmen L; Voronine, Dmitri V

2018-05-03

Optical detection and conformational mapping of aptamers are important for improving medical and biosensing technologies and for better understanding of biological processes at the molecular level. The authors investigate the vibrational signals of deoxyribonucleic acid aptamers specific to Listeria monocytogenes immobilized on gold substrates using tip-enhanced Raman scattering (TERS) spectroscopy and nanoscale imaging. The authors compare topographic and nano-optical signals and investigate the fluctuations of the position-dependent TERS spectra. They perform spatial TERS mapping with 3 nm step size and discuss the limitation of the resulting spatial resolution under the ambient conditions. TERS mapping provides information about the chemical composition and conformation of aptamers and paves the way to future label-free biosensing.

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.

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

PubMed Central

Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

2013-01-01

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

Atomic Layer Deposition of Wet-Etch Resistant Silicon Nitride Using Di(sec-butylamino)silane and N2 Plasma on Planar and 3D Substrate Topographies.

PubMed

Faraz, Tahsin; van Drunen, Maarten; Knoops, Harm C M; Mallikarjunan, Anupama; Buchanan, Iain; Hausmann, Dennis M; Henri, Jon; Kessels, Wilhelmus M M

2017-01-18

The advent of three-dimensional (3D) finFET transistors and emergence of novel memory technologies place stringent requirements on the processing of silicon nitride (SiN x ) films used for a variety of applications in device manufacturing. In many cases, a low temperature (<400 °C) deposition process is desired that yields high quality SiN x films that are etch resistant and also conformal when grown on 3D substrate topographies. In this work, we developed a novel plasma-enhanced atomic layer deposition (PEALD) process for SiN x using a mono-aminosilane precursor, di(sec-butylamino)silane (DSBAS, SiH 3 N( s Bu) 2 ), and N 2 plasma. Material properties have been analyzed over a wide stage temperature range (100-500 °C) and compared with those obtained in our previous work for SiN x deposited using a bis-aminosilane precursor, bis(tert-butylamino)silane (BTBAS, SiH 2 (NH t Bu) 2 ), and N 2 plasma. Dense films (∼3.1 g/cm 3 ) with low C, O, and H contents at low substrate temperatures (<400 °C) were obtained on planar substrates for this process when compared to other processes reported in the literature. The developed process was also used for depositing SiN x films on high aspect ratio (4.5:1) 3D trench nanostructures to investigate film conformality and wet-etch resistance (in dilute hydrofluoric acid, HF/H 2 O = 1:100) relevant for state-of-the-art device architectures. Film conformality was below the desired levels of >95% and attributed to the combined role played by nitrogen plasma soft saturation, radical species recombination, and ion directionality during SiN x deposition on 3D substrates. Yet, very low wet-etch rates (WER ≤ 2 nm/min) were observed at the top, sidewall, and bottom trench regions of the most conformal film deposited at low substrate temperature (<400 °C), which confirmed that the process is applicable for depositing high quality SiN x films on both planar and 3D substrate topographies.

Enhanced Light Extraction from OLEDs Fabricated on Patterned Plastic Substrates

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

Hippola, Chamika; Kaudal, Rajiv; Manna, Eeshita

A key scientific and technological challenge in organic light-emitting diodes (OLEDs) is enhancing the light outcoupling factor η out, which is typically <20%. This paper reports experimental and modeling results of a promising approach to strongly increase η out by fabricating OLEDs on novel flexible nanopatterned substrates that result in a >2× enhancement in green phosphorescent OLEDs (PhOLEDs) fabricated on corrugated polycarbonate (PC). The external quantum efficiency (EQE) reaches 50% (meaning ηout ≥50%); it increases 2.6x relative to a glass/ITO device and 2× relative to devices on glass/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) or flat PC/PEDOT:PSS. A significant enhancement is also observed formore » blue PhOLEDs with EQE 1.7× relative to flat PC. The corrugated PC substrates are fabricated efficiently and cost-effectively by direct room-temperature molding. These substrates successfully reduce photon losses due to trapping/waveguiding in the organic+anode layers and possibly substrate, and losses to plasmons at the metal cathode. Focused ion beam gauged the conformality of the OLEDs. Dome-shaped convex nanopatterns with height of ~280–400 nm and pitch ~750–800 nm were found to be optimal. Lastly, substrate design and layer thickness simulations, reported first for patterned devices, agree with the experimental results that present a promising method to mitigate photon loss paths in OLEDs.« less

Enhanced Light Extraction from OLEDs Fabricated on Patterned Plastic Substrates

DOE PAGES

Hippola, Chamika; Kaudal, Rajiv; Manna, Eeshita; ...

2018-02-19

A key scientific and technological challenge in organic light-emitting diodes (OLEDs) is enhancing the light outcoupling factor η out, which is typically <20%. This paper reports experimental and modeling results of a promising approach to strongly increase η out by fabricating OLEDs on novel flexible nanopatterned substrates that result in a >2× enhancement in green phosphorescent OLEDs (PhOLEDs) fabricated on corrugated polycarbonate (PC). The external quantum efficiency (EQE) reaches 50% (meaning ηout ≥50%); it increases 2.6x relative to a glass/ITO device and 2× relative to devices on glass/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) or flat PC/PEDOT:PSS. A significant enhancement is also observed formore » blue PhOLEDs with EQE 1.7× relative to flat PC. The corrugated PC substrates are fabricated efficiently and cost-effectively by direct room-temperature molding. These substrates successfully reduce photon losses due to trapping/waveguiding in the organic+anode layers and possibly substrate, and losses to plasmons at the metal cathode. Focused ion beam gauged the conformality of the OLEDs. Dome-shaped convex nanopatterns with height of ~280–400 nm and pitch ~750–800 nm were found to be optimal. Lastly, substrate design and layer thickness simulations, reported first for patterned devices, agree with the experimental results that present a promising method to mitigate photon loss paths in OLEDs.« less

Fabrication of textured SnO2 transparent conductive films using self-assembled Sn nanospheres

NASA Astrophysics Data System (ADS)

Fukumoto, Michitaka; Nakao, Shoichiro; Hirose, Yasushi; Hasegawa, Tetsuya

2018-06-01

We present a novel method to fabricate textured surfaces on transparent conductive SnO2 films by processing substrates through a bottom-up technique with potential for industrially scalable production. The substrate processing consists of three steps: deposition of precursor Sn films on glass substrates, formation of a self-assembled Sn nanosphere layer with reductive annealing, and conversion of Sn to SnO2 by oxidative annealing. Ta-doped SnO2 films conformally deposited on the self-assembled nanospherical SnO2 templates exhibited attractive optical and electrical properties, namely, enhanced haze values and low sheet resistances, for applications as transparent electrodes in photovoltaics.

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

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

PubMed

Banerjee, Rupa; Jayaraj, Gopal Gunanathan; Peter, Joshua Jebakumar; Kumar, Vignesh; Mapa, Koyeli

2016-08-01

DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures. © 2016 Federation of European Biochemical Societies.

Epoxide Hydrolase Conformational Heterogeneity for the Resolution of Bulky Pharmacologically Relevant Epoxide Substrates.

PubMed

Serrano-Hervás, Eila; Casadevall, Guillem; Garcia-Borràs, Marc; Feixas, Ferran; Osuna, Sílvia

2018-04-06

The conformational landscape of Bacillus megaterium epoxide hydrolase (BmEH) and how it is altered by mutations that confer the enzyme the ability to accept bulky epoxide substrates has been investigated. Extensive molecular dynamics (MD) simulations coupled to active site volume calculations have unveiled relevant features of the enzyme conformational dynamics and function. Our long-timescale MD simulations identify key conformational states not previously observed by means of X-ray crystallography and short MD simulations that present the loop containing one of the catalytic residues, Asp239, in a wide-open conformation, which is likely involved in the binding of the epoxide substrate. Introduction of mutations M145S and F128A dramatically alters the conformational landscape of the enzyme. These singly mutated variants can accept bulky epoxide substrates due to the disorder induced by mutation in the α-helix containing the catalytic Tyr144 and some parts of the lid domain. These changes impact the enzyme active site, which is substantially wider and more complementary to the bulky pharmacologically relevant epoxide substrates. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

PubMed Central

Kirmizialtin, Serdal; Nguyen, Virginia; Johnson, Kenneth A.; Elber, Ron

2012-01-01

Summary Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate. PMID:22483109

Following the crowd: Brain Substrates of Long-Term Memory Conformity

PubMed Central

Edelson, Micah; Sharot, Tali; Dolan, Raymond J; Dudai, Yadin

2012-01-01

Human memory is strikingly susceptible to social influences, yet we know little about the underlying mechanisms. We examined how socially induced memory errors are generated in the brain by studying the memory of individuals exposed to recollections of others. Participants exhibited a strong tendency to conform to erroneous recollections of the group, producing both long-lasting and temporary errors, even when their initial memory was strong and accurate. Functional brain imaging revealed that social influence modified the neuronal representation of memory. Specifically, a particular brain signature of enhanced amygdala activity and enhanced amygdala-hippocampus connectivity predicted long-lasting, but not temporary memory alterations. Our findings reveal how social manipulation can alter memory and extend the known functions of the amygdala to encompass socially-mediated memory distortions. PMID:21719681

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.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration.

PubMed

Stewart, Barry J; Wardle, Simon J; Haniford, David B

2002-08-15

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

PubMed Central

Stewart, Barry J.; Wardle, Simon J.; Haniford, David B.

2002-01-01

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes. PMID:12169640

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

The properties and performance of moisture/oxygen barrier layers deposited by remote plasma sputtering

NASA Astrophysics Data System (ADS)

Brown, Hayley Louise

The development of flexible lightweight OLED devices requires oxygen/moisture barrier layer thin films with water vapour transmission rates (WVTR) of < 10-6 g/m2/day. This thesis reports on single and multilayer architecture barrier layers (mostly based on SiO2, Al2O3 and TiO2) deposited onto glass, Si and polymeric substrates using remote plasma sputtering. The reactive sputtering depositions were performed on Plasma Quest S500 based sputter systems and the morphology, nanostructure and composition of the coatings have been examined using SEM, EDX, STEM, XPS, XRD and AFM. The WVTR has been determined using industry standard techniques (e.g. MOCON) but, for rapid screening of the deposited layers, an in-house permeation test was also developed. SEM, XRD and STEM results showed that the coatings exhibited a dense, amorphous structure with no evidence of columnar growth. However, all of the single and multilayer coatings exhibited relatively poor WVTRs of > 1 x 10-1 g/m2/day at 38 °C and 85 % RH. Further characterisation indicated that the barrier films were failing due to the presence of substrate asperities and airborne particulates. Different mechanisms were investigated in an attempt to reduce the density of film defects including incorporation of a getter layer, modification of growth kinetics, plasma treatment and polymer planarising, but none were successful in lowering the WVTR. Review of this issue indicated that the achievement of good barrier layers was likely to be problematic in commercial practice due to the cost implications of adequately reducing particulate density and the need to cover deliberately non-planar surfaces and fabricated 3D structures. Conformal coverage would therefore be required to bury surface structures and to mitigate particulate issues. Studies of the remote plasma system showed that it both inherently delivered an ionised physical vapour deposition (IPVD) process and was compatible with bias re-sputtering of substrates. Accordingly, a process using RF substrate bias to conformally coat surfaces was developed to encapsulate surface particulates and seal associated permeation paths. An order of magnitude improvement in WVTR (6.7 x 10-2 g/m2/day) was measured for initial Al2O3 coatings deposited with substrate bias. The development of substrate bias to enhance conformal coverage provides significant new commercial benefit. Furthermore, conformal coverage of 5:1 aspect ratio structures have been demonstrated by alternating the substrate bias between -222 V and -267 V, with a 50 % dwell time at each voltage. Further development and optimisation of the substrate bias technique is required to fully explore the potential for further improving barrier properties and conformal coverage of high aspect ratio and other 3D structures.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Noninvasive noble metal nanoparticle arrays for surface-enhanced Raman spectroscopy of proteins

NASA Astrophysics Data System (ADS)

Inya-Agha, Obianuju; Forster, Robert J.; Keyes, Tia E.

2007-02-01

Noble metal nanoparticles arrays are well established substrates for surface enhanced Raman spectroscopy (SERS). Their ability to enhance optical fields is based on the interaction of their surface valence electrons with incident electromagnetic radiation. In the array configuration, noble metal nanoparticles have been used to produce SER spectral enhancements of up to 10 8 orders of magnitude, making them useful for the trace analysis of physiologically relevant analytes such as proteins and peptides. Electrostatic interactions between proteins and metal surfaces result in the preferential adsorption of positively charged protein domains onto metal surfaces. This preferential interaction has the effect of disrupting the native conformation of the protein fold, with a concomitant loss of protein function. A major historic advantage of Raman microspectroscopy has been is its non-invasive nature; protein denaturation on the metal surfaces required for SER spectroscopy renders it a much more invasive technique. Further, part of the analytical power of Raman spectroscopy lies in its use as a secondary conformation probe. The protein structural loss which occurs on the metal surface results in secondary conformation readings which are not true to the actual native state of the analyte. This work presents a method for chemical fabrication of noble metal SERS arrays with surface immobilized layers which can protect protein native conformation without excessively mitigating the electromagnetic enhancements of spectra. Peptide analytes are used as model systems for proteins. Raman spectra of alpha lactalbumin on surfaces and when immobilized on these novel arrays are compared. We discuss the ability of the surface layer to protect protein structure whilst improving signal intensity.

Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin.

PubMed

Idakieva, Krassimira; Siddiqui, Nurul Islam; Meersman, Filip; De Maeyer, Marc; Chakarska, Irena; Gielens, Constant

2009-08-01

The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by limited treatment with subtilisin and by incubation of RtH with 2.9 mM sodium dodecyl sulphate (SDS), suggesting an enhanced substrate access to the active sites. The highest artificial induction of o-diPO activity in RtH, however, was obtained by lyophilization of the protein. This is ascribed to conformational changes during the lyophilization process of the didecameric RtH molecules, affecting the accessibility of the active sites. These conformational changes must be very small, since Fourier-transform infrared and circular dichroism spectroscopies did not reveal any changes in secondary structure of lyophilized RtH. The difference in accessibility of the copper containing active site for substrates between catechol oxidase and functional unit RtH2-e was demonstrated by molecular modeling and surface area accessibility calculations. The low level of intrinsic PO activity in the investigated hemocyanin is related to the inaccessibility of the binuclear copper active sites to the substrates.

Conformational dynamics of the molecular chaperone Hsp90

PubMed Central

Krukenberg, Kristin A.; Street, Timothy O.; Lavery, Laura A.; Agard, David A.

2016-01-01

The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1–2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organism-dependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90’s conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90’s conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics. PMID:21414251

Self-assembled diatom substrates with plasmonic functionality

NASA Astrophysics Data System (ADS)

Kwon, Sun Yong; Park, Sehyun; Nichols, William T.

2014-04-01

Marine diatoms have an exquisitely complex exoskeleton that is promising for engineered surfaces such as sensors and catalysts. For such applications, creating uniform arrays of diatom frustules across centimeter scales will be necessary. Here, we present a simple, low-cost floating interface technique to self-assemble the diatom frustules. We show that well-prepared diatoms form floating hexagonal close-packed arrays at the air-water interface that can be transferred directly to a substrate. We functionalize the assembled diatom surfaces with gold and characterize the plasmonic functionality by using surface enhanced Raman scattering (SERS). Thin gold films conform to the complex, hierarchical diatom structure and produce a SERS enhancement factor of 2 × 104. Small gold nanoparticles attached to the diatom's surface produce a higher enhancement of 7 × 104 due to stronger localization of the surface plasmons. Taken together, the large-scale assembly and plasmonic functionalization represent a promising platform to control the energy and the material flows at a complex surface for applications such as sensors and plasmonic enhanced catalysts.

Plasmonic Paper as a Novel Chem/Bio Detection Platform

NASA Astrophysics Data System (ADS)

Tian, Limei

The time varying electric field of electromagnetic (EM) radiation causes oscillation of conduction electrons of metal nanoparticles. The resonance of such oscillation, termed localized surface plasmon resonance (LSPR), falls into the visible spectral region for noble metals such as gold, silver and copper. LSPR of metal nanostructures is sensitive to numerous factors such as composition, size, shape, dielectric properties of surrounding medium, and proximity to other nanostructures (plasmon coupling). The sensitivity of LSPR to the refractive index of surrounding medium renders it an attractive platform for chemical and biological sensing. When the excitation light is in resonance with the plasmon frequency of the metal nanoparticle, it radiates a characteristic dipolar radiation causing a characteristic spatial distribution in which certain areas show higher EM field intensity, which is manifested as electromagnetic field enhancement. Surface enhanced Raman scattering (SERS) involves dramatic enhancement of the intensity of the Raman scattering from the analyte adsorbed on or in proximity to a nanostructured metal surface exhibiting such strong EM field enhancement. Both LSPR and SERS have been widely investigated for highly sensitive and label-free chemical & biological sensors. Most of the SERS/LSPR sensors demonstrated so far rely on rigid planar substrates (e.g., glass, silicon) owing to the well-established lithographic approaches, which are routinely employed for either fabrication or assembly of plasmonic nanotransducers. In many cases, their rigid nature results in low conformal contact with the sample and hence poor sample collection efficiency. We hypothesized that paper substrates are an excellent alternative to conventional rigid substrates to significantly improve the (multi-)functionality of LSPR/SERS substrates, dramatically simplify the fabrication procedures and lower the cost. The choice of paper substrates for the implementation of SERS/LSPR sensors is rationalized by numerous advantages such as (i) high specific surface area resulting in large dynamic range (ii) excellent wicking properties for rapid uptake and transport of analytes to test domains (iii) compatibility with conventional printing approaches, enabling multi-analyte plasmonic sensors (iv) significant reduction in cost (v) smaller sample volume requirement (vi) easy disposability. In this work, we have introduced novel SERS and LSPR substrates based on conventional filter paper decorated with plasmonic nanostructures, called plasmonic paper. A flexible SERS substrate based on common filter paper adsorbed with gold nanostructures allows conformal contact with real-world surfaces, enabling rapid trace detection. To realize multifunctional SERS substrates, paper substrates were cut into star-shaped structures and the fingers were differentially functionalized with polyelectrolytes that allows separation and pre-concentration of different components of a complex sample in a small surface area by taking advantage of the properties of cellulose paper and shape-enhanced capillary effect. Plasmonic paper can also serve as a novel LSPR biosensing platform by decorating the paper substrate with biofunctionalized nanostructures. Furthermore, calligraphy approach was employed to create well-isolated test domains on paper substrates using functionalized plasmonic nanostructures as ink for multiplexed chemical sensing and label-free biosensing. These plasmonic paper substrates exhibit excellent sample collection efficiency and do not require complex fabrication processes. This class of substrates is expected to have applications not only to first responders and military personal but also to several areas of medical, food analysis, and environmental research.

Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

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

S Vaidya; E Velazquez-Delgado; G Abbruzzese

2011-12-31

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergomore » a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.« less

Substrate bias induced synthesis of flowered-like bunched carbon nanotube directly on bulk nickel

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

Bisht, Atul; Academy of Scientific and Innovative Research; Chockalingam, S.

2016-02-15

Highlights: • Flowered-like bunched MWCNTs have been synthesized by MW PECVD technique. • Effect of substrate bias on the properties of MWCNT has been studied. • Minimum E{sub T} = 1.9 V/μm with β = 4770 has been obtained in the film deposited at −350 V. - Abstract: This paper reports the effect of substrate bias on the multiwalled carbon nanotube (MWCNT) deposited on nickel foil by microwave plasma enhanced chemical vapor deposition technique. The MWCNTs have been characterized by the scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), Raman spectroscopy, field emission and current–voltage characteristic of themore » heterojunction diode. The SEM images exhibit unique hierarchical flowered-like bunched and conformally coated MWCNTs. Substrate bias induced ion bombardment helps in the enhancement of hydrocarbon dissociation and is responsible for flowered-like MWCNTs growth. The HRTEM micrographs show the base growth mechanism for MWCNTs. The value of turn on field for emission decreases from 5.5 to 1.9 V/μm and field enhancement factor increases from 927 to 4770, respectively, with the increase of substrate bias. The diode ideality factor of CNT/ n-Si heterojunction is evaluated as 2.4 and the on/off current ratio is found to be 7 at ±2 V, respectively.« less

Statistical properties of fluctuating enzymes with dynamic cooperativity using a first passage time distribution formalism.

PubMed

Singh, Divya; Chaudhury, Srabanti

2017-04-14

We study the temporal fluctuations in catalytic rates for single enzyme reactions undergoing slow transitions between two active states. We use a first passage time distribution formalism to obtain the closed-form analytical expressions of the mean reaction time and the randomness parameter for reaction schemes where conformational fluctuations are present between two free enzyme conformers. Our studies confirm that the sole presence of free enzyme fluctuations yields a non Michaelis-Menten equation and can lead to dynamic cooperativity. The randomness parameter, which is a measure of the dynamic disorder in the system, converges to unity at a high substrate concentration. If slow fluctuations are present between the enzyme-substrate conformers (off-pathway mechanism), dynamic disorder is present at a high substrate concentration. Our results confirm that the dynamic disorder at a high substrate concentration is determined only by the slow fluctuations between the enzyme-substrate conformers and the randomness parameter is greater than unity. Slow conformational fluctuations between free enzymes are responsible for the emergence of dynamic cooperativity in single enzymes. Our theoretical findings are well supported by comparison with experimental data on the single enzyme beta-galactosidase.

DNA origami based Au-Ag-core-shell nanoparticle dimers with single-molecule SERS sensitivity

NASA Astrophysics Data System (ADS)

Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.; Bald, I.

2016-03-01

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA bands, SEM images, additional AFM images, FDTD simulations, additional reference spectra for Cy3 and detailed description of EF estimation, simulated absorption and scattering spectra. See DOI: 10.1039/c5nr08674d

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

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.

Effect of divalent ions on the optical emission behavior of protein thin films

NASA Astrophysics Data System (ADS)

Bhowal, Ashim Chandra; Kundu, Sarathi

2016-05-01

Photoluminescence behaviors of proteinthin film, bovine serum albumin (BSA) have been studied in the presence of three divalent ions (Mg2+, Ca2+ and Ba2+) at different temperatures using fluorescence spectroscopy. Film thickness and morphology have been studied using atomic force microscopy. Variation of different physicochemical parameters like temperature, solvent polarity, pH, ionic strength, substrate binding etc. can make conformational changes in the protein structure and hence influences the emission behavior.In thin film conformation of BSA, dynamic quenching behavior has beenidentified in the presence of all the three divalent ions at pH≈ 5.5. Depending upon the charge density of the divalent ions interaction with protein molecules modifies and as a result quenching efficiency varies. Also after heat treatment, conformation of the protein molecules changes and as a result the quenching efficiency enhances than that of the unheated films. Studies on such protein-ion interactions and conformational variation may explore various functions of protein when it will adsorb on soft surfaces like membranes, vesicles, etc.

Plasma Amino Acid Coatings for a Conformal Growth of Titania Nanoparticles

DTIC Science & Technology

2010-04-01

of dry nitrogen. Periodic porous polymer SU8 templates fabricated using multi-beam IL according to published procedures have been supplied by Thomas...3D periodic polymer structures via plasma enhanced chemical vapor deposition. We demonstrate the efficient utilization of this functional amino acid...nanoparticles were grown directly on histidine-functionalized planar and 3D polymer substrates by a wet-chemistry method that showed uniform surface

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

PubMed Central

Jagtap, Pravin Kumar Ankush; Soni, Vijay; Vithani, Neha; Jhingan, Gagan Deep; Bais, Vaibhav Singh; Nandicoori, Vinay Kumar; Prakash, Balaji

2012-01-01

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU. PMID:22969087

Thermoelectric material including conformal oxide layers and method of making the same using atomic layer deposition

DOEpatents

Cho, Jung Young; Ahn, Dongjoon; Salvador, James R.; Meisner, Gregory P.

2016-06-07

A thermoelectric material includes a substrate particle and a plurality of conformal oxide layers formed on the substrate particle. The plurality of conformal oxide layers has a total oxide layer thickness ranging from about 2 nm to about 20 nm. The thermoelectric material excludes oxide nanoparticles. A method of making the thermoelectric material is also disclosed herein.

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.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

PubMed

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Salt bridge dynamics control substrate-induced conformational change in the membrane transporter GlpT

PubMed Central

Law, Christopher J.; Almqvist, Jonas; Bernstein, Adam; Goetz, Regina M.; Huang, Yafei; Soudant, Celine; Laaksonen, Aatto; Hovmöller, Sven; Wang, Da-Neng

2008-01-01

Summary Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (Pi) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate/phosphate antiporters. PMID:18395745

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

PubMed

Brewer, J M; Wampler, J E

2001-03-14

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Relating conformation to function in integrin α5β1.

PubMed

Su, Yang; Xia, Wei; Li, Jing; Walz, Thomas; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

2016-07-05

Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

Trade-off between Photon Management Efficacy and Material Quality in Thin-Film Solar Cells on Nanostructured Substrates of High Aspect Ratio Structures

DOE PAGES

Chin, Alan; Keshavarz, Majid; Wang, Qi

2018-04-13

Although texturing of the transparent electrode of thin-film solar cells has long been used to enhance light absorption via light trapping, such texturing has involved low aspect ratio features. With the recent development of nanotechnology, nanostructured substrates enable improved light trapping and enhanced optical absorption via resonances, a process known as photon management, in thin-film solar cells. Despite the progress made in the development of photon management in thin-film solar cells using nanostructures substrates, the structural integrity of the thin-film solar cells deposited onto such nanostructured substrates is rarely considered. Here, we report the observation of the reduction in themore » open circuit voltage of amorphous silicon solar cells deposited onto a nanostructured substrate with increasing areal number density of high aspect ratio structures. For a nanostructured substrate with the areal number density of such nanostructures increasing in correlation with the distance from one edge of the substrate, a correlation between the open circuit voltage reduction and the increase of the areal number density of high aspect ratio nanostructures of the front electrode of the small-size amorphous silicon solar cells deposited onto different regions of the substrate with graded nanostructure density indicates the effect of the surface morphology on the material quality, i.e., a trade-off between photon management efficacy and material quality. Lastly, this observed trade-off highlights the importance of optimizing the morphology of the nanostructured substrate to ensure conformal deposition of the thin-film solar cell.« less

Trade-off between Photon Management Efficacy and Material Quality in Thin-Film Solar Cells on Nanostructured Substrates of High Aspect Ratio Structures

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

Chin, Alan; Keshavarz, Majid; Wang, Qi

Although texturing of the transparent electrode of thin-film solar cells has long been used to enhance light absorption via light trapping, such texturing has involved low aspect ratio features. With the recent development of nanotechnology, nanostructured substrates enable improved light trapping and enhanced optical absorption via resonances, a process known as photon management, in thin-film solar cells. Despite the progress made in the development of photon management in thin-film solar cells using nanostructures substrates, the structural integrity of the thin-film solar cells deposited onto such nanostructured substrates is rarely considered. Here, we report the observation of the reduction in themore » open circuit voltage of amorphous silicon solar cells deposited onto a nanostructured substrate with increasing areal number density of high aspect ratio structures. For a nanostructured substrate with the areal number density of such nanostructures increasing in correlation with the distance from one edge of the substrate, a correlation between the open circuit voltage reduction and the increase of the areal number density of high aspect ratio nanostructures of the front electrode of the small-size amorphous silicon solar cells deposited onto different regions of the substrate with graded nanostructure density indicates the effect of the surface morphology on the material quality, i.e., a trade-off between photon management efficacy and material quality. Lastly, this observed trade-off highlights the importance of optimizing the morphology of the nanostructured substrate to ensure conformal deposition of the thin-film solar cell.« less

Conformational Dynamics of a Y-Family DNA Polymerase during Substrate Binding and Catalysis As Revealed by Interdomain Förster Resonance Energy Transfer

PubMed Central

2015-01-01

Numerous kinetic, structural, and theoretical studies have established that DNA polymerases adjust their domain structures to enclose nucleotides in their active sites and then rearrange critical active site residues and substrates for catalysis, with the latter conformational change acting to kinetically limit the correct nucleotide incorporation rate. Additionally, structural studies have revealed a large conformational change between the apoprotein and the DNA–protein binary state for Y-family DNA polymerases. In previous studies [Xu, C., Maxwell, B. A., Brown, J. A., Zhang, L., and Suo, Z. (2009) PLoS Biol.7, e1000225], a real-time Förster resonance energy transfer (FRET) method was developed to monitor the global conformational transitions of DNA polymerase IV from Sulfolobus solfataricus (Dpo4), a prototype Y-family enzyme, during nucleotide binding and incorporation by measuring changes in distance between locations on the enzyme and the DNA substrate. To elucidate further details of the conformational transitions of Dpo4 during substrate binding and catalysis, in this study, the real-time FRET technique was used to monitor changes in distance between various pairs of locations in the protein itself. In addition to providing new insight into the conformational changes as revealed in previous studies, the results here show that the previously described conformational change between the apo and DNA-bound states of Dpo4 occurs in a mechanistic step distinct from initial formation or dissociation of the binary complex of Dpo4 and DNA. PMID:24568554

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

PubMed Central

Dastvan, Reza; Mishra, Smriti; Meiler, Jens; Mchaourab, Hassane S.

2016-01-01

The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1–3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient. PMID:26787875

A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

PubMed Central

Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

2015-01-01

Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

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.

Enhanced piezoelectricity and stretchability in energy harvesting devices fabricated from buckled PZT ribbons.

PubMed

Qi, Yi; Kim, Jihoon; Nguyen, Thanh D; Lisko, Bozhena; Purohit, Prashant K; McAlpine, Michael C

2011-03-09

The development of a method for integrating highly efficient energy conversion materials onto soft, biocompatible substrates could yield breakthroughs in implantable or wearable energy harvesting systems. Of particular interest are devices which can conform to irregular, curved surfaces, and operate in vital environments that may involve both flexing and stretching modes. Previous studies have shown significant advances in the integration of highly efficient piezoelectric nanocrystals on flexible and bendable substrates. Yet, such inorganic nanomaterials are mechanically incompatible with the extreme elasticity of elastomeric substrates. Here, we present a novel strategy for overcoming these limitations, by generating wavy piezoelectric ribbons on silicone rubber. Our results show that the amplitudes in the waves accommodate order-of-magnitude increases in maximum tensile strain without fracture. Further, local probing of the buckled ribbons reveals an enhancement in the piezoelectric effect of up to 70%, thus representing the highest reported piezoelectric response on a stretchable medium. These results allow for the integration of energy conversion devices which operate in stretching mode via reversible deformations in the wavy/buckled ribbons.

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

PubMed Central

Grouleff, Julie; Schiøtt, Birgit

2013-01-01

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design. PMID:23776432

Plasma-enhanced atomic layer deposition for plasmonic TiN

NASA Astrophysics Data System (ADS)

Otto, Lauren M.; Hammack, Aaron T.; Aloni, Shaul; Ogletree, D. Frank; Olynick, Deirdre L.; Dhuey, Scott; Stadler, Bethanie J. H.; Schwartzberg, Adam M.

2016-09-01

This work presents the low temperature plasma-enhanced atomic layer deposition (PE-ALD) of TiN, a promising plasmonic synthetic metal. The plasmonics community has immediate needs for alternatives to traditional plasmonic materials (e.g. Ag and Au), which lack chemical, thermal, and mechanical stability. Plasmonic alloys and synthetic metals have significantly improved stability, but their growth can require high-temperatures (>400 °C), and it is difficult to control the thickness and directionality of the resulting film, especially on technologically important substrates. Such issues prevent the application of alternative plasmonic materials for both fundamental studies and large-scale industrial applications. Alternatively, PE-ALD allows for conformal deposition on a variety of substrates with consistent material properties. This conformal coating will allow the creation of exotic three-dimensional structures, and low-temperature deposition techniques will provide unrestricted usage across a variety of platforms. The characterization of this new plasmonic material was performed with in-situ spectroscopic ellipsometry as well as Auger electron spectroscopy for analysis of TiN film sensitivity to oxide cross-contamination. Plasmonic TiN films were fabricated, and a chlorine plasma etch was found to pattern two dimensional gratings as a test structure. Optical measurements of 900 nm period gratings showed reasonable agreement with theoretical modeling of the fabricated structures, indicating that ellipsometry models of the TiN were indeed accurate.

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

Conformational Changes Allow Processing of Bulky Substrates by a Haloalkane Dehalogenase with a Small and Buried Active Site.

PubMed

Kokkonen, Piia; Bednar, David; Dockalova, Veronika; Prokop, Zbynek; Damborsky, Jiri

2018-06-01

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and which is likely the route for bulky substrates. These results provide insights into the structure-dynamics-function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to DCE introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Conformable amplified lead zirconate titanate sensors with enhanced piezoelectric response for cutaneous pressure monitoring.

PubMed

Dagdeviren, Canan; Su, Yewang; Joe, Pauline; Yona, Raissa; Liu, Yuhao; Kim, Yun-Soung; Huang, YongAn; Damadoran, Anoop R; Xia, Jing; Martin, Lane W; Huang, Yonggang; Rogers, John A

2014-08-05

The ability to measure subtle changes in arterial pressure using devices mounted on the skin can be valuable for monitoring vital signs in emergency care, detecting the early onset of cardiovascular disease and continuously assessing health status. Conventional technologies are well suited for use in traditional clinical settings, but cannot be easily adapted for sustained use during daily activities. Here we introduce a conformal device that avoids these limitations. Ultrathin inorganic piezoelectric and semiconductor materials on elastomer substrates enable amplified, low hysteresis measurements of pressure on the skin, with high levels of sensitivity (~0.005 Pa) and fast response times (~0.1 ms). Experimental and theoretical studies reveal enhanced piezoelectric responses in lead zirconate titanate that follow from integration on soft supports as well as engineering behaviours of the associated devices. Calibrated measurements of pressure variations of blood flow in near-surface arteries demonstrate capabilities for measuring radial artery augmentation index and pulse pressure velocity.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

PubMed

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-05-29

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

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

Direct Growth of Graphene Films on 3D Grating Structural Quartz Substrates for High-Performance Pressure-Sensitive Sensors.

PubMed

Song, Xuefen; Sun, Tai; Yang, Jun; Yu, Leyong; Wei, Dacheng; Fang, Liang; Lu, Bin; Du, Chunlei; Wei, Dapeng

2016-07-06

Conformal graphene films have directly been synthesized on the surface of grating microstructured quartz substrates by a simple chemical vapor deposition process. The wonderful conformality and relatively high quality of the as-prepared graphene on the three-dimensional substrate have been verified by scanning electron microscopy and Raman spectra. This conformal graphene film possesses excellent electrical and optical properties with a sheet resistance of <2000 Ω·sq(-1) and a transmittance of >80% (at 550 nm), which can be attached with a flat graphene film on a poly(dimethylsiloxane) substrate, and then could work as a pressure-sensitive sensor. This device possesses a high-pressure sensitivity of -6.524 kPa(-1) in a low-pressure range of 0-200 Pa. Meanwhile, this pressure-sensitive sensor exhibits super-reliability (≥5000 cycles) and an ultrafast response time (≤4 ms). Owing to these features, this pressure-sensitive sensor based on 3D conformal graphene is adequately introduced to test wind pressure, expressing higher accuracy and a lower background noise level than a market anemometer.

Enzyme Architecture: Erection of Active Orotidine 5'-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes.

PubMed

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

2017-11-15

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with k cat /K m = 1.4 × 10 -7 M -1 s -1 . Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.

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.

Role of conformational dynamics in the evolution of novel enzyme function.

PubMed

Maria-Solano, Miguel A; Serrano-Hervás, Eila; Romero-Rivera, Adrian; Iglesias-Fernández, Javier; Osuna, Sílvia

2018-05-21

The free energy landscape concept that describes enzymes as an ensemble of differently populated conformational sub-states in dynamic equilibrium is key for evaluating enzyme activity, enantioselectivity, and specificity. Mutations introduced in the enzyme sequence can alter the populations of the pre-existing conformational states, thus strongly modifying the enzyme ability to accommodate alternative substrates, revert its enantiopreferences, and even increase the activity for some residual promiscuous reactions. In this feature article, we present an overview of the current experimental and computational strategies to explore the conformational free energy landscape of enzymes. We provide a series of recent publications that highlight the key role of conformational dynamics for the enzyme evolution towards new functions and substrates, and provide some perspectives on how conformational dynamism should be considered in future computational enzyme design protocols.

Plasma enhanced chemical vapour deposition of silica onto Ti: Analysis of surface chemistry, morphology and functional hydroxyl groups

PubMed Central

Szili, Endre J.; Kumar, Sunil; Smart, Roger St. C.; Lowe, Rachel; Saiz, Eduardo; Voelcker, Nicolas H.

2009-01-01

Previously, we have developed and characterised a procedure for the deposition of thin silica films by a plasma enhanced chemical vapour deposition (PECVD) procedure using tetraethoxysilane (TEOS) as the main precursor. We have used the silica coatings for improving the corrosion resistance of metals and for enhancing the bioactivity of biomedical metallic implants. Recently, we have been fine-tuning the PECVD method for producing high quality and reproducible PECVD-silica (PECVD-Si) coatings on metals, primarily for biomaterial applications. In order to understand the interaction of the PECVD-Si coatings with biological species (such as proteins and cells), it is important to first analyse the properties of the silica films deposited using the optimised parameters. Therefore, this current investigation was carried out to analyse the characteristic features of PECVD-Si deposited on Ti substrates (PECVD-Si-Ti). We determined that the PECVD-Si coatings on Ti were conformal to the substrate surface, strongly adhered to the underlying substrate and were resistant to delamination. The PECVD-Si surface was composed of stoichiometric SiO2, showed a low carbon content (below 10 at.%) and was very hydrophilic (contact angle <10°). Finally, we also showed that the PECVD-Si coatings contain functional hydroxyl groups. PMID:19809536

White thin-film flip-chip LEDs with uniform color temperature using laser lift-off and conformal phosphor coating technologies.

PubMed

Lin, Huan-Ting; Tien, Ching-Ho; Hsu, Chen-Peng; Horng, Ray-Hua

2014-12-29

We fabricated a phosphor-conversion white light emitting diode (PC-WLED) using a thin-film flip-chip GaN LED with a roughened u-GaN surface (TFFC-SR-LED) that emits blue light at 450 nm wavelength with a conformal phosphor coating that converts the blue light into yellow light. It was found that the TFFC-SR-LED with the thin-film substrate removal process and surface roughening exhibits a power enhancement of 16.1% when compared with the TFFC-LED without a sapphire substrate. When a TFFC-SR-LED with phosphors on a Cu-metal packaging-base (TFFC-SR-Cu-WLED) was operated at a forward-bias current of 350 mA, luminous flux and luminous efficacy were increased by 17.8 and 11.9%, compared to a TFFC-SR-LED on a Cup-shaped packaging-base (TFFC-SR-Cup-WLED). The angular correlated color temperature (CCT) deviation of a TFFC-SR-Cu-WLED reaches 77 K in the range of -70° to + 70° when the average CCT of white LEDs is around 4300 K. Consequently, the TFFC-SR-LED in a conformal coating phosphor structure on a Cu packaging-base could not only increase the luminous flux output, but also improve the angular-dependent CCT uniformity, thereby reducing the yellow ring effect.

Development of Advanced Conformal Ablative TPS Fabricated from Rayon- and PAN-Based Carbon Felts

NASA Technical Reports Server (NTRS)

Gasch, Matthew; Stackpoole, Margaret; White, Susan; Boghozian, Tane

2016-01-01

The conformal ablative TPS first developed under NASA's Hypersonics Project in the early 2000's demonstrated very low through the thickness conductivity compared to state-ofthe- art PICA. However, in initial arcjet testing of Conformal-1, surface recession rates were 2x higher than PICA. Because commercial carbon felts are currently available as very thin substrates, this was a concern if conformal TPS were to be considered for a mission that required thicker material. Discussed in this paper are the results of the development of an Advanced Conformal TPS derived from thicker, higher density carbon felt. Two substrate systems were evaluated, the first material was a needled rayon-based carbon felt and the other a needled PAN-based carbon felt. Both substrates were impregnated with phenolic resin following the PICA/CPICA process to add a low density phenolic matrix to the system prior to aerothermal screening at the LaRC HyMETS facility and larger scale testing in the NASA ARC Interaction Heating Facility (IHF) at heating fluxes ranging from 250-1700 W/cm2.

Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release

PubMed Central

Koldsø, Heidi; Noer, Pernille; Grouleff, Julie; Autzen, Henriette Elisabeth; Sinning, Steffen; Schiøtt, Birgit

2011-01-01

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport. PMID:22046120

Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions

PubMed Central

Rekha, Nambudiry; Srinivasan, N

2003-01-01

Background Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (α) and two identical regulatory (β) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2α in isolation. The present work explores the structural basis of this feature using computational analysis and modeling. Results We have initially built a model of PKCK2α bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2α and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in β turn, α helix, or 310 helices. This type of conformation is observed and accommodated well by PKCK2α in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2α phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2α complex. PKCK2β undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme. Conclusion Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2α, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme. PMID:12740046

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

PubMed Central

Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

2013-01-01

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

Design and analysis of a conformal patch antenna for a wearable breast hyperthermia treatment system

NASA Astrophysics Data System (ADS)

Curto, Sergio; Ramasamy, Manoshika; Suh, Minyoung; Prakash, Punit

2015-03-01

To overcome the limitations of currently available clinical hyperthermia systems which are based on rigid waveguide antennas, a wearable microwave hyperthermia system is presented. A light wearable system can improve patient comfort and be located in close proximity to the breast, thereby enhancing energy deposition and reducing power requirements. The objective of this work was to design and assess the feasibility of a conformal patch antenna element of an array system to be integrated into a wearable hyperthermia bra. The feasibility of implementing antennas with silver printed ink technology on flexible substrates was evaluated. A coupled electromagnetic-bioheat transfer solver and a hemispheric heterogeneous numerical breast phantom were used to design and optimize a 915 MHz patch antenna. The optimization goals were device miniaturization, operating bandwidth, enhanced energy deposition pattern in targets, and reduced Efield back radiation. The antenna performance was evaluated for devices incorporating a hemispheric conformal groundplane and a rectangular groundplane configuration. Simulated results indicated a stable -10 dB return loss bandwidth of 88 MHz for both the conformal and rectangular groundplane configurations. Considering applied power levels restricted to 15 W, treatment volumes (T>410C) and depth from the skin surface were 11.32 cm3 and 27.94 mm, respectively, for the conformal groundplane configuration, and 2.79 cm3 and 19.72 mm, respectively, for the rectangular groundplane configuration. E-field back-radiation reduced by 85.06% for the conformal groundplane compared to the rectangular groundplane configuration. A prototype antenna with rectangular groundplane was fabricatd and experimentally evaluated. The groundplane was created by printing silver ink (Metalon JS-B25P) on polyethylene terephthalate (PET) film surface. Experiments revealed stable antenna performance for power levels up to 15.3 W. In conclusion, the proposed patch antenna with conformal groundplane and prined ink technology shows promising performance to be integrated in a clinical array system.

Origins of stereoselectivity in evolved ketoreductases.

PubMed

Noey, Elizabeth L; Tibrewal, Nidhi; Jiménez-Osés, Gonzalo; Osuna, Sílvia; Park, Jiyong; Bond, Carly M; Cascio, Duilio; Liang, Jack; Zhang, Xiyun; Huisman, Gjalt W; Tang, Yi; Houk, Kendall N

2015-12-22

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Origins of stereoselectivity in evolved ketoreductases

PubMed Central

Noey, Elizabeth L.; Tibrewal, Nidhi; Jiménez-Osés, Gonzalo; Osuna, Sílvia; Park, Jiyong; Bond, Carly M.; Cascio, Duilio; Liang, Jack; Zhang, Xiyun; Huisman, Gjalt W.; Tang, Yi; Houk, Kendall N.

2015-01-01

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP+-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation. PMID:26644568

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.

A Peptide/MHCII conformer generated in the presence of exchange peptide is substrate for HLA-DM editing

PubMed Central

Ferrante, Andrea; Gorski, Jack

2012-01-01

The mechanism of HLA-DM (DM) activity is still unclear. We have shown that DM-mediated peptide release from HLA-DR (DR) is dependent on the presence of exchange peptide. However, DM also promotes a small amount of peptide release in the absence of exchange peptide. Here we show that SDS-PAGE separates purified peptide/DR1 complexes (pDR1) into two conformers whose ratio is peptide Kd-dependent. In the absence of exchange peptide, DM only releases peptide from the slower migrating conformer. Addition of exchange peptide converts the DM-resistant conformer to the slower migrating conformer, which is DM labile. Thus, exchange peptide generates a conformer of pDR1 which constitutes the intermediate for peptide exchange and the substrate for DM activity. The resolution of the intermediate favors the highest affinity peptide. However, once folded into the DM-resistant conformer, even low affinity peptides can be presented in the absence of free peptide, broadening the repertoire available for presentation. PMID:22545194

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

PubMed Central

Santoso, Yusdi; Joyce, Catherine M.; Potapova, Olga; Le Reste, Ludovic; Hohlbein, Johannes; Torella, Joseph P.; Grindley, Nigel D. F.; Kapanidis, Achillefs N.

2010-01-01

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates. PMID:20080740

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Energy level alignment and molecular conformation at rubrene/Ag interfaces: Impact of contact contaminations on the interfaces

NASA Astrophysics Data System (ADS)

Sinha, Sumona; Wang, C.-H.; Mukherjee, M.

2017-07-01

This paper addresses the impact of electrode contaminations on the interfacial energy level alignment, the molecular conformation, orientation and surface morphology deposited organic film at organic semiconductor/noble metal interfaces by varying of film thickness from sub-monolayer to multilayer, which currently draws significant attention with regard to its application in organic electronics. The UHV clean Ag and unclean Ag were employed as substrate whereas rubrene was used as an organic semiconducting material. The photoelectron spectroscopy (XPS and UPS) was engaged to investigate the evolution of interfacial energetics; polarization dependent near edge x-ray absorption fine structure spectroscopy (NEXAFS) was employed to understand the molecular conformation as well as orientation whereas atomic force microscopy (AFM) was used to investigate the surface morphologies of the films. The adventitious contamination layer was acted as a spacer layer between clean Ag substrate surface and rubrene molecular layer. As a consequence, hole injection barrier height, interface dipole as well as molecular-conformation, molecular-orientation and surface morphology of rubrene thin films were found to depend on the cleanliness of Ag substrate. The results have important inferences about the understanding of the impact of substrate contamination on the energy level alignment, the molecular conformation as well as orientation and surface morphology of deposited rubrene thin film at rubrene/Ag interfaces and are beneficial for the improvement of the device performance.

Conformal Robotic Stereolithography

PubMed Central

Stevens, Adam G.; Oliver, C. Ryan; Kirchmeyer, Matthieu; Wu, Jieyuan; Chin, Lillian; Polsen, Erik S.; Archer, Chad; Boyle, Casey; Garber, Jenna

2016-01-01

Abstract Additive manufacturing by layerwise photopolymerization, commonly called stereolithography (SLA), is attractive due to its high resolution and diversity of materials chemistry. However, traditional SLA methods are restricted to planar substrates and planar layers that are perpendicular to a single-axis build direction. Here, we present a robotic system that is capable of maskless layerwise photopolymerization on curved surfaces, enabling production of large-area conformal patterns and the construction of conformal freeform objects. The system comprises an industrial six-axis robot and a custom-built maskless projector end effector. Use of the system involves creating a mesh representation of the freeform substrate, generation of a triangulated toolpath with curved layers that represents the target object to be printed, precision mounting of the substrate in the robot workspace, and robotic photopatterning of the target object by coordinated motion of the robot and substrate. We demonstrate printing of conformal photopatterns on spheres of various sizes, and construction of miniature three-dimensional objects on spheres without requiring support features. Improvement of the motion accuracy and development of freeform toolpaths would enable construction of polymer objects that surpass the size and support structure constraints imparted by traditional SLA systems. PMID:29577062

The Structural Basis of ATP as an Allosteric Modulator

PubMed Central

Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

2014-01-01

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

Electrically responsive microstructured polypyrrole-polyurethane composites for stimulated osteogenesis

NASA Astrophysics Data System (ADS)

Luculescu, Catalin Romeo; Acasandrei, Adriana Maria; Mustaciosu, Cosmin Catalin; Zamfirescu, Marian; Dinescu, Maria; Calin, Bogdan Stefanita; Popescu, Andrei; Chioibasu, Diana; Cristian, Dan; Paun, Irina Alexandra

2018-03-01

In this work, we demonstrate the efficiency of substrate-mediated electrical stimulation of micropatterned polypyrrole/polyurethane (PPy/PU) composites for enhancing the osteogenesis in osteoblast-like cells. The PPy/PU substrates were obtained by dispersing electrically conductive PPy nanograins within a mechanically resistant PU matrix. Spin-coated PPy/PU layers were micropatterned with predefined 3D geometries by ultrashort laser ablation. Then they were conformally coated by Matrix Assisted Pulsed Laser Evaporation, in order to restore their chemical and electrical integrity. The chemical structure of the laser-processed PPy/PU substrates was investigated by 2D and 3D mapping of the laser-processed areas, via Raman microspectroscopy. In vitro studies revealed that the micropatterned PPy/PU substrates facilitated the topological and electrical communication of the seeded osteoblasts. Specifically, we demonstrated the cells attachment on the predefined 3D micropatterns. More importantly, we found evidence about the cells mineralization inside the 3D micropatterns by investigating the calcium deposits by Energy-Dispersive X-Ray Spectroscopy (EDS) and Alizarin Red staining. We found that the substrate-mediated electrical stimulation of the PPy/PU substrates induced a twofold increase of the Ca deposits in the cultured cells.

The effects of nanophase ceramic materials on select functions of human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Dulgar-Tulloch, Aaron Joseph

2005-11-01

Modification of the chemistry and surface topography of nanophase ceramics can provide biomaterial formulations capable of directing the functions of adherent cells. This effect relies on the type, amount, and conformation of adsorbed proteins that mediate the adhesion of mesenchymally-descended lineages. The mechanisms driving this response are not yet well-understood and have not been investigated for human mesenchymal stem cells (HMSCs), a progenitor-lineage critical to orthopaedic biomaterials. The present study addressed these needs by examining the in vitro adhesion, proliferation, and osteogenic differentiation of HMSCs as a function of substrate chemistry and grain size, with particular attention to the protein-mediated mechanisms of cell adhesion. Alumina, titania, and hydroxyapatite substrates were prepared with 1500, 200, 50, and 24 (alumina only) nm grain sizes, and characterized with respect to surface properties, porosity, composition, and phase. Adhesion of HMSCs was dependent upon both chemistry and grain size. Specifically, adhesion on alumina and hydroxyapatite was reduced on 50 and 24 (alumina only) nm surfaces, as compared to 1500 and 200 nm surfaces, while adhesion on titania substrates was independent of grain size. Investigation into the protein-mediated mechanisms of this response identified vitronectin as the dominant adhesive protein, demonstrated random protein distribution across the substrate surface without aggregation or segregation, and confirmed the importance of the type, amount, and conformation of adsorbed proteins in cell adhesion. Minimal cell proliferation was observed on 50 and 24 (alumina only) nm substrates of any chemistry. Furthermore, cell proliferation was up-regulated on 200 nm substrates after 7 days of culture. Osteogenic differentiation was not detected on 50 nm substrates throughout the 28 day culture period. In contrast, osteogenic differentiation was strongly enhanced on 200 nm substrates, occurring approximately 7 days earlier and in greater magnitude than that observed on 1500 nm substrates. In summary, the current study elucidated the chemical and topographical cues necessary to optimize the vitronectin-mediated adhesion, proliferation, and differentiation of human mesenchymal stem cells on ceramic surfaces. These results expand the understanding of surface-mediated cell functions and provide information pertinent to the design of next-generation orthopaedic and tissue engineering biomaterials.

CONTRASTING BEHAVIOR OF CONFORMATIONALLY LOCKED CARBOCYCLIC NUCLEOSIDES OF ADENOSINE AND CYTIDINE AS SUBSTRATES FOR DEAMINASES

PubMed Central

Schroeder, Gottfried K.; Ludek, Olaf R.; Siddiqui, Maqbool A.; Ezzitouni, Abdallah; Wolfenden, Richard

2010-01-01

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn+2 coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4’) oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold. PMID:20183605

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.

The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate.

PubMed Central

Roberts, D; Kellett, G L

1979-01-01

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state. Images Fig. 5. Fig. 8. PMID:160791

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

PubMed

Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

2017-06-06

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

PubMed Central

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-01-01

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions. PMID:23624933

Conformal dip-coating of patterned surfaces for capillary die-to-substrate self-assembly

NASA Astrophysics Data System (ADS)

Mastrangeli, M.; Ruythooren, W.; Van Hoof, C.; Celis, J.-P.

2009-04-01

Capillarity-driven self-assembly of small chips onto planar target substrates is a promising alternative to robotic pick-and-place assembly. It critically relies on the selective deposition of thin fluid films on patterned binding sites, which is anyway normally non-conformal. We found that the addition of a thin wetting sidewall, surrounding the entire site perimeter, enables the conformal fluid coverage of arbitrarily shaped sites through dip-coating, significantly improves the reproducibility of the coating process and strongly reduces its sensitivity to surface defects. In this paper we support the feasibility and potential of this method by demonstrating the conformal dip-coating of square and triangular sites conditioned with combinations of different hydrophobic and hydrophilic surface chemistries. We present both experimental and simulative evidence of the advantages brought by the introduction of the wetting boundary on film coverage accuracy. Application of our surface preparation method to capillary self-assembly could result in higher precision in die-to-substrate registration and larger freedom in site shape design.

Conformally encapsulated multi-electrode arrays with seamless insulation

DOEpatents

Tabada, Phillipe J.; Shah, Kedar G.; Tolosa, Vanessa; Pannu, Satinderall S.; Tooker, Angela; Delima, Terri; Sheth, Heeral; Felix, Sarah

2016-11-22

Thin-film multi-electrode arrays (MEA) having one or more electrically conductive beams conformally encapsulated in a seamless block of electrically insulating material, and methods of fabricating such MEAs using reproducible, microfabrication processes. One or more electrically conductive traces are formed on scaffold material that is subsequently removed to suspend the traces over a substrate by support portions of the trace beam in contact with the substrate. By encapsulating the suspended traces, either individually or together, with a single continuous layer of an electrically insulating material, a seamless block of electrically insulating material is formed that conforms to the shape of the trace beam structure, including any trace backings which provide suspension support. Electrical contacts, electrodes, or leads of the traces are exposed from the encapsulated trace beam structure by removing the substrate.

Appendage mountable electronic devices conformable to surfaces

DOEpatents

Rogers, John; Ying, Ming; Bonifas, Andrew; Lu, Nanshu

2017-01-24

Disclosed are appendage mountable electronic systems and related methods for covering and conforming to an appendage surface. A flexible or stretchable substrate has an inner surface for receiving an appendage, including an appendage having a curved surface, and an opposed outer surface that is accessible to external surfaces. A stretchable or flexible electronic device is supported by the substrate inner and/or outer surface, depending on the application of interest. The electronic device in combination with the substrate provides a net bending stiffness to facilitate conformal contact between the inner surface and a surface of the appendage provided within the enclosure. In an aspect, the system is capable of surface flipping without adversely impacting electronic device functionality, such as electronic devices comprising arrays of sensors, actuators, or both sensors and actuators.

A multifunctional biphasic water splitting catalyst tailored for integration with high-performance semiconductor photoanodes

NASA Astrophysics Data System (ADS)

Yang, Jinhui; Cooper, Jason K.; Toma, Francesca M.; Walczak, Karl A.; Favaro, Marco; Beeman, Jeffrey W.; Hess, Lucas H.; Wang, Cheng; Zhu, Chenhui; Gul, Sheraz; Yano, Junko; Kisielowski, Christian; Schwartzberg, Adam; Sharp, Ian D.

2017-03-01

Artificial photosystems are advanced by the development of conformal catalytic materials that promote desired chemical transformations, while also maintaining stability and minimizing parasitic light absorption for integration on surfaces of semiconductor light absorbers. Here, we demonstrate that multifunctional, nanoscale catalysts that enable high-performance photoelectrochemical energy conversion can be engineered by plasma-enhanced atomic layer deposition. The collective properties of tailored Co3O4/Co(OH)2 thin films simultaneously provide high activity for water splitting, permit efficient interfacial charge transport from semiconductor substrates, and enhance durability of chemically sensitive interfaces. These films comprise compact and continuous nanocrystalline Co3O4 spinel that is impervious to phase transformation and impermeable to ions, thereby providing effective protection of the underlying substrate. Moreover, a secondary phase of structurally disordered and chemically labile Co(OH)2 is introduced to ensure a high concentration of catalytically active sites. Application of this coating to photovoltaic p+n-Si junctions yields best reported performance characteristics for crystalline Si photoanodes.

Substrate uptake and protein stability relationship in mammalian histidine decarboxylase.

PubMed

Pino-Angeles, A; Morreale, A; Negri, A; Sánchez-Jiménez, F; Moya-García, A A

2010-01-01

There is some evidence linking the substrate entrance in the active site of mammalian histidine decarboxylase and an increased stability against proteolytic degradation. In this work, we study the basis of this relationship by means of protein structure network analysis and molecular dynamics simulations. We find that the substrate binding to the active site influences the conformation of a flexible region sensible to proteolytic degradation and observe how formation of the Michaelis-Menten complex increases stability in the conformation of this region. (c) 2009 Wiley-Liss, Inc.

Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

PubMed

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

2016-08-22

Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

NASA Astrophysics Data System (ADS)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

PubMed Central

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

PubMed

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

2016-02-10

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Illuminating the Mechanistic Roles of Enzyme Conformational Dynamics

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

Hanson, Jeffrey A.; Dunderstadt, Karl; Watkins, Lucas P.

2007-11-13

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme’s lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK’s domain movements on its catalytic time scale. To quantitatively measure the enzyme’s entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly,more » the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme’s conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme’s rate-limiting step.« less

Performance Evaluation of Strain Gauge Printed Using Automatic Fluid Dispensing System on Conformal Substrates

NASA Astrophysics Data System (ADS)

Khairilhijra Khirotdin, Rd.; Faridzuan Ngadiron, Mohamad; Adzeem Mahadzir, Muhammad; Hassan, Nurhafizzah

2017-08-01

Smart textiles require flexible electronics that can withstand daily stresses like bends and stretches. Printing using conductive inks provides the flexibility required but the current printing techniques suffered from ink incompatibility, limited of substrates to be printed with and incompatible with conformal substrates due to its rigidity and low flexibility. An alternate printing technique via automatic fluid dispensing system is proposed and its performances on printing strain gauge on conformal substrates were evaluated to determine its feasibility. Process parameters studied including printing speed, deposition height, curing time and curing temperature. It was found that the strain gauge is proven functional as expected since different strains were induced when bent on variation of bending angles and curvature radiuses from designated bending fixtures. The average change of resistances were doubled before the strain gauge starts to break. Printed strain gauges also exhibited some excellence elasticity as they were able to resist bending up to 70° angle and 3 mm of curvature radius.

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate.

PubMed

Zuniga, Jorge E; Schmidt, James J; Fenn, Timothy; Burnett, James C; Araç, Demet; Gussio, Rick; Stafford, Robert G; Badie, Shirin S; Bavari, Sina; Brunger, Axel T

2008-10-08

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.

Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing.

PubMed

Snoberger, Aaron; Brettrager, Evan J; Smith, David M

2018-06-18

Protein degradation in all domains of life requires ATPases that unfold and inject proteins into compartmentalized proteolytic chambers. Proteasomal ATPases in eukaryotes and archaea contain poorly understood N-terminally conserved coiled-coil domains. In this study, we engineer disulfide crosslinks in the coiled-coils of the archaeal proteasomal ATPase (PAN) and report that its three identical coiled-coil domains can adopt three different conformations: (1) in-register and zipped, (2) in-register and partially unzipped, and (3) out-of-register. This conformational heterogeneity conflicts with PAN's symmetrical OB-coiled-coil crystal structure but resembles the conformational heterogeneity of the 26S proteasomal ATPases' coiled-coils. Furthermore, we find that one coiled-coil can be conformationally constrained even while unfolding substrates, and conformational changes in two of the coiled-coils regulate PAN switching between resting and active states. This switching functionally mimics similar states proposed for the 26S proteasome from cryo-EM. These findings thus build a mechanistic framework to understand regulation of proteasome activity.

Substrate Control in Stereoselective Lanthionine Biosynthesis

PubMed Central

Tang, Weixin; Jiménez-Osés, Gonzalo; Houk, K. N.; van der Donk, Wilfred A.

2014-01-01

Enzymes are typically highly stereoselective catalysts that enforce a reactive conformation on their native substrates. We report here a rare example where the substrate controls the stereoselectivity of an enzyme-catalyzed Michael-type addition during the biosynthesis of lanthipeptides. These natural products contain thioether crosslinks formed by cysteine attack on dehydrated Ser and Thr residues. We demonstrate that several lanthionine synthetases catalyze highly selective anti additions in which the substrate (and not the enzyme) determines whether the addition occurs from the Re or Si face. A single point mutation in the peptide substrate completely inverted the stereochemical outcome of the enzymatic modification. Quantum mechanical calculations reproduced the experimentally observed selectivity and suggest that conformational restraints imposed by the amino acid sequence on the transition states determine the face selectivity of the Michael-type cyclization. PMID:25515891

Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

PubMed Central

Yeung, Heidi O.; Förster, Andreas; Bebeacua, Cecilia; Niwa, Hajime; Ewens, Caroline; McKeown, Ciarán; Zhang, Xiaodong; Freemont, Paul S.

2014-01-01

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes. PMID:24598262

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

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

On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

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

Brokaw, Jason B.; Chu, Jhih-wei

2010-11-17

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier worksmore » of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.« less

Methods of manipulating stressed epistructures

DOEpatents

Wanlass, Mark W

2014-04-08

A method of processing an epistructure or processing a semiconductor device including associating a conformal and flexible handle with the epistructure and removing the epistructure and handle as a unit from the parent substrate. The method further includes causing the epistructure and handle unit to conform to a shape that differs from the shape the epistructure otherwise inherently assumes upon removal from the parent substrate. A device prepared according to the disclosed methods.

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates

PubMed Central

Bhat, Shreyas; Hasenhuetl, Peter S.; Kasture, Ameya; El-Kasaby, Ali; Baumann, Michael H.; Blough, Bruce E.; Sucic, Sonja; Sandtner, Walter; Freissmuth, Michael

2017-01-01

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to μm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants. PMID:28842491

Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

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

Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth

2009-09-11

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft aremore » more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.« less

Hydrophobically stabilized open state for the lateral gate of the Sec translocon

PubMed Central

Zhang, Bin; Miller, Thomas F.

2010-01-01

The Sec translocon is a central component of cellular pathways for protein translocation and membrane integration. Using both atomistic and coarse-grained molecular simulations, we investigate the conformational landscape of the translocon and explore the role of peptide substrates in the regulation of the translocation and integration pathways. Inclusion of a hydrophobic peptide substrate in the translocon stabilizes the opening of the lateral gate for membrane integration, whereas a hydrophilic peptide substrate favors the closed lateral gate conformation. The relative orientation of the plug moiety and a peptide substrate within the translocon channel is similarly dependent on whether the substrate is hydrophobic or hydrophilic in character, and the energetics of the translocon lateral gate opening in the presence of a peptide substrate is governed by the energetics of the peptide interface with the membrane. Implications of these results for the regulation of Sec-mediated pathways for protein translocation vs. membrane integration are discussed. PMID:20203009

X-ray structures of LeuT in substrate-free outward-open and apo inward-open states

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

Krishnamurthy, Harini; Gouaux, Eric

2012-08-09

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA ({gamma}-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes themore » extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.« less

SERS substrates for in-situ biosensing (Conference Presentation)

NASA Astrophysics Data System (ADS)

Venugopalan, Priyamvada; Quilis, Nestor; Jakub, Dostalek; Wolfgang, Knoll

2017-06-01

Abstract: Recent years have seen a rapid progress in the field of surface-enhanced Raman spectroscopy (SERS) which is attributed to the thriving field of plasmonics [1]. SERS is a susceptible technique that can address basic scientific questions and technological problems. In both cases, it is highly dependent upon the plasmonic substrate, where excitation of the localized surface plasmon resonance enhances the vibrational scattering signal of the analyte molecules adsorbed on to the surface [2]. In this work, using finite difference time domain (FDTD) method we investigate the optical properties of plasmonic nanostructures with tuned plasmonic resonances as a function of dielectric environment and geometric parameters. An optimized geometry will be discussed based on the plasmonic resonant position and the SERS intensity. These SERS substrates will be employed for the detection of changes in conformation caused by interactions between an aptamer and analyte molecules. This will be done by using a microfluidic channel designed within the configuration of the lab-on-a-chip concept based on the intensity changes of the SERS signal. More efficient and reproducible results are obtained for such a quantitative measurement of analytes at low concentration levels. We will also demonstrate that the plasmonic substrates fabricated by top down approach such as e-beam lithography (EBL) and laser interference lithography (LIL) are highly reproducible, robust and can result in high electric field enhancement. Our results demonstrate the potential to use SERS substrates for highly sensitive detection schemes opening up the window for a wide range of applications including biomedical diagnostics, forensic investigation etc. Acknowledgement: This work was supported by the Austrian Science Fund (FWF), project NANOBIOSENSOR (I 2647). References: [1] J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao and R. P. V. Duyne., " Biosensing with plasmonic nanosensors," Nature materials, 308(7), 2008. [2] T. Y. Jeon1, D. J. Kim, S. Park, S. Kim and D. Kim., "Nanostructured plasmonic substrates for use as SERS sensors," Nanocovergence, 3(18), 2016.

Computational Insights into an Enzyme-Catalyzed [4+2] Cycloaddition

PubMed Central

2017-01-01

The enzyme SpnF, involved in the biosynthesis of spinosyn A, catalyzes a formal [4+2] cycloaddition of a 22-membered macrolactone, which may proceed as a concerted [4+2] Diels–Alder reaction or a stepwise [6+4] cycloaddition followed by a Cope rearrangement. Quantum mechanics/molecular mechanics (QM/MM) calculations combined with free energy simulations show that the Diels–Alder pathway is favored in the enzyme environment. OM2/CHARMM free energy simulations for the SpnF-catalyzed reaction predict a free energy barrier of 22 kcal/mol for the concerted Diels–Alder process and provide no evidence of a competitive stepwise pathway. Compared with the gas phase, the enzyme lowers the Diels–Alder barrier significantly, consistent with experimental observations. Inspection of the optimized geometries indicates that the enzyme may prearrange the substrate within the active site to accelerate the [4+2] cycloaddition and impede the [6+4] cycloaddition through interactions with active-site residues. Judging from partial charge analysis, we find that the hydrogen bond between the Thr196 residue of SpnF and the substrate C15 carbonyl group contributes to the enhancement of the rate of the Diels–Alder reaction. QM/MM simulations show that the substrate can easily adopt a reactive conformation in the active site of SpnF because interconversion between the C5–C6 s-trans and s-cis conformers is facile. Our QM/MM study suggests that the enzyme SpnF does behave as a Diels-Alderase. PMID:29131960

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

NASA Astrophysics Data System (ADS)

Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

2016-04-01

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

Thermally evaporated conformal thin films on non-traditional/non-planar substrates

NASA Astrophysics Data System (ADS)

Pulsifer, Drew Patrick

Conformal thin films have a wide variety of uses in the microelectronics, optics, and coatings industries. The ever-increasing capabilities of these conformal thin films have enabled tremendous technological advancement in the last half century. During this period, new thin-film deposition techniques have been developed and refined. While these techniques have remarkable performance for traditional applications which utilize planar substrates such as silicon wafers, they are not suitable for the conformal coating of non-traditional substrates such as biological material. The process of thermally evaporating a material under vacuum conditions is one of the oldest thin-film deposition techniques which is able to produce functional film morphologies. A drawback of thermally evaporated thin films is that they are not intrinsically conformal. To overcome this, while maintaining the advantages of thermal evaporation, a procedure for varying the substrates orientation with respect to the incident vapor flux during deposition was developed immediately prior to the research undertaken for this doctoral dissertation. This process was shown to greatly improve the conformality of thermally evaporated thin films. This development allows for several applications of thermally evaporated conformal thin films on non-planar/non-traditional substrates. Three settings in which to evaluate the improved conformal deposition of thermally evaporated thin films were investigated for this dissertation. In these settings the thin-film morphologies are of different types. In the first setting, a bioreplication approach was used to fabricate artificial visual decoys for the invasive species Agrilus planipennis, commonly known as the emerald ash borer (EAB). The mating behavior of this species involves an overflying EAB male pouncing on an EAB female at rest on an ash leaflet before copulation. The male spots the female on the leaflet by visually detecting the iridescent green color of the female's elytra. As rearing EAB and then deploying dead females as decoys is both arduous and inconvenient, the development of an artificial decoy would be of great interest to entomologists and foresters. A dead female EAB was used to make a negative die of nickel and a positive die of epoxy. The process of fabricating the paired dies utilized thermally evaporated conformal thin films in several critical steps. In order to conformally coat the EAB with nickel, the substrate stage holding the female EAB was periodically rocked and rotated during the deposition. This process was designed to result in a uniform thin film of ˜ 500-nm thickness with dense morphology. The nickel film was then reinforced through an electroforming process and mounted in a fixture which allowed it to be heated electrically. The corresponding positive die was replicated from the negative die through a series of successive castings. The final EAB positive die was fabricated from a hard epoxy material and attached to a fixture which allowed it to be heated while being pressed into the negative die. Decoys were then made by first depositing a quarter-wave-stack Bragg reflector on a polymer sheet and then stamping it with the pair of matched negative and positive dies to take the shape of the upper surface of an EAB female. As nearly 100 decoys were fabricated from just one EAB female, this bioreplication process is industrially scalable. Preliminary results from field trapping tests are indicative of success. For the second setting, a method of developing latent fingermarks with thermally evaporated conformal thin films was developed. Fingermarks have long been used to identify the individual who left them behind when he/she touched an object with the friction ridges of his/her hands. In many cases the fingermark which is left behind consists of sebaceous secretions which are not clearly visible under normal conditions. In order to make the fingermarks visible and identifiable, they are traditionally developed by either a physical technique which relies on a material preferentially sticking to sebaceous materials or a chemical technique which relies on a reaction with material within the fingermark. In this application, a columnar thin film (CTF) is deposited conformally over both the fingermark and the underlying substrate. The CTF is produced by the conformal-evaporated-film-by-rotation method, wherein the substrate with the fingermark upon it is held obliquely with respect to a vapor flux in a vacuum chamber. The substrate is then rapidly rotated about its surface normal resulting in a conformal film with columnar morphology. This technique was optimized for several substrates and compared with traditional development techniques. CTF development was found to be superior to traditional techniques in several cases. Use of the CTF was investigated for several types of particularly difficult to develop fingermarks such as those which consist of both bloody and nonbloody areas, and fingermarks on fired cartridge casings. The CTF technique's sensitivity was also compared to that of traditional development techniques. Finally, the CTF technique was compared with another thin film deposition technique called vacuum-metal deposition. (Abstract shortened by UMI.).

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase.

PubMed

Lee, Chi Chung; Fay, Aaron W; Weng, Tsu-Chien; Krest, Courtney M; Hedman, Britt; Hodgson, Keith O; Hu, Yilin; Ribbe, Markus W

2015-11-10

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

PubMed Central

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

PubMed

Kumar, Ramasamy P; Morehouse, Benjamin R; Matos, Jason O; Malik, Karan; Lin, Hongkun; Krauss, Isaac J; Oprian, Daniel D

2017-03-28

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K I values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K I values are similar to the K M for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k cat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s -1 , respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

PubMed Central

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2013-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth. PMID:23527883

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

PubMed

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2014-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

Nanoporous films: From conventional to the conformal

DOE PAGES

Allendorf, Mark D.; Stavila, Vitalie

2015-12-14

Here, thin and continuous films of porous metal-organic frameworks can now be conformally deposited on various substrates using a vapor-phase synthesis approach that departs from conventional solution-based routes.

Movie of the structural changes during a catalytic cycle of nucleoside monophosphate kinases.

PubMed

Vonrhein, C; Schlauderer, G J; Schulz, G E

1995-05-15

There are 17 crystal structures of nucleoside monophosphate kinases known. As expected for kinases, they show large conformational changes upon binding of substrates. These are concentrated in two chain segments, or domains, of 30 and 38 residues that are involved in binding of the substrates N1TP and N2MP (nucleoside tri- and monophosphates with bases N1 and N2), respectively. After aligning the 17 structures on the main parts of their polypeptide chains, two domains in various conformational states were revealed. These states were caused by bound substrate (or analogues) and by crystal-packing forces, and ranged between a 'closed' conformation and a less well defined 'open' conformation. The structures were visually sorted yielding an approximately evenly spaced series of domain states that outlines the closing motions when the substrates bind. The packing forces in the crystals are weak, leaving the natural domain trajectories essentially intact. Packing is necessary, however, to produce stable intermediates. The ordered experimental structures were then recorded as still pictures of a movie and animated to represent the motions of the molecule during a catalytic cycle. The motions were smoothed out by adding interpolated structures to the observed ones. The resulting movies are available through the World Wide Web (http:@bio5.chemie.uni-freiburg.de/ak movie.html). Given the proliferating number of homologous proteins known to exist in different conformational states, it is becoming possible to outline the motions of chain segments and combine them into a movie, which can then represent protein action much more effectively than static pictures alone are able to do.

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.

Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli.

PubMed Central

Pomeranz Krummel, D A; Altman, S

1999-01-01

M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta[273-281]), but not for the M1 RNA-product complex. High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex. PMID:10445877

Porous light-emitting compositions

DOEpatents

Burrell, Anthony K [Los Alamos, NM; McCleskey, Thomas Mark [Los Alamos, NM; Jia, Quanxi [Los Alamos, NM; Bauer, Eve [Los Alamos, NM; Mueller, Alexander H [Los Alamos, NM

2012-04-17

Light-emitting devices are prepared by coating a porous substrate using a polymer-assisted deposition process. Solutions of metal precursor and soluble polymers having binding properties for metal precursor were coated onto porous substrates. The coated substrates were heated at high temperatures under a suitable atmosphere. The result was a substrate with a conformal coating that did not substantially block the pores of the substrate.

Techniques used to study the DNA polymerase reaction pathway

PubMed Central

Joyce, Catherine M.

2009-01-01

Summary A minimal reaction pathway for DNA polymerases was established over 20 years ago using chemical quench methods. Since that time there has been considerable interest in noncovalent steps in the reaction pathway, conformational changes involving the polymerase or its DNA substrate that may play a role in substrate specificity. Fluorescence-based assays have been devised in order to study these conformational transitions and the results obtained have added new detail to the reaction pathway. PMID:19665596

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

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

A multifunctional biphasic water splitting catalyst tailored for integration with high-performance semiconductor photoanodes

DOE PAGES

Yang, Jinhui; Cooper, Jason K.; Toma, Francesca M.; ...

2016-11-07

Artificial photosystems are advanced by the development of conformal catalytic materials that promote desired chemical transformations, while also maintaining stability and minimizing parasitic light absorption for integration on surfaces of semiconductor light absorbers. We demonstrate that multifunctional, nanoscale catalysts that enable high-performance photoelectrochemical energy conversion can be engineered by plasma-enhanced atomic layer deposition. The collective properties of tailored Co 3 O 4 /Co(OH) 2 thin films simultaneously provide high activity for water splitting, permit efficient interfacial charge transport from semiconductor substrates, and enhance durability of chemically sensitive interfaces. Furthermore, these films comprise compact and continuous nanocrystalline Co 3 O 4more » spinel that is impervious to phase transformation and impermeable to ions, thereby providing effective protection of the underlying substrate. Moreover, a secondary phase of structurally disordered and chemically labile Co(OH) 2 is introduced to ensure a high concentration of catalytically active sites. Application of this coating to photovoltaic p + n-Si junctions yields best reported performance characteristics for crystalline Si photoanodes.« less

The Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding†

PubMed Central

Clift, Michael; Ji, Haitao; Deniau, Gildas P.; O’Hagan, David; Silverman, Richard B.

2008-01-01

γ-Aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5’-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5’-phosphate (PLP) to pyridoxamine 5’-phosphate (PMP). The enzyme then catalyzes the conversion of α-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred based on the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches. PMID:17988152

Induced-fit Mechanism for Prolyl Endopeptidase

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

Li, Min; Chen, Changqing; Davies, David R.

2010-11-15

Prolyl peptidases cleave proteins at proline residues and are of importance for cancer, neurological function, and type II diabetes. Prolyl endopeptidase (PEP) cleaves neuropeptides and is a drug target for neuropsychiatric diseases such as post-traumatic stress disorder, depression, and schizophrenia. Previous structural analyses showing little differences between native and substrate-bound structures have suggested a lock-and-key catalytic mechanism. We now directly demonstrate from seven structures of Aeromonus punctata PEP that the mechanism is instead induced fit: the native enzyme exists in a conformationally flexible opened state with a large interdomain opening between the {beta}-propeller and {alpha}/{beta}-hydrolase domains; addition of substrate tomore » preformed native crystals induces a large scale conformational change into a closed state with induced-fit adjustments of the active site, and inhibition of this conformational change prevents substrate binding. Absolute sequence conservation among 28 orthologs of residues at the active site and critical residues at the interdomain interface indicates that this mechanism is conserved in all PEPs. This finding has immediate implications for the use of conformationally targeted drug design to improve specificity of inhibition against this family of proline-specific serine proteases.« less

Modulation of Cell Proliferation and Differentiation through Substrate-dependent Changes in Fibronectin Conformation

PubMed Central

García, Andrés J.; Vega, María D.; Boettiger, David

1999-01-01

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound α5 and β1 integrin subunits but not αv or β3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of α5β1 integrin bound to Fn, and differentiation was inhibited by anti-α5, but not anti-αv, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications. PMID:10069818

Conformational change of adenosine deaminase during ligand-exchange in a crystal.

PubMed

Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

2008-08-15

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

Transverse ac-driven and geometric ratchet effects for vortices in conformal crystal pinning arrays

DOE PAGES

Reichhardt, Charles; Reichhardt, Cynthia Jane Olsen

2016-02-11

A conformal pinning array is created by taking a conformal transformation of a uniform hexagonal lattice to create a structure in which the sixfold ordering of the original lattice is preserved but which has a spatial gradient in the pinning site density. With a series of conformal arrays it is possible to create asymmetric substrates, and it was previously shown that when an ac drive is applied parallel to the asymmetry direction, a pronounced ratchet effect occurs with a net dc flow of vortices in the same direction as the ac drive. Here, in this article, we show that whenmore » the ac drive is applied perpendicular to the substrate asymmetry direction, it is possible to realize a transverse ratchet effect where a net dc flow of vortices is generated perpendicular to the ac drive. The conformal transverse ratchet effect is distinct from previous versions of transverse ratchets in that it occurs due to the generation of non-Gaussian transverse vortex velocity fluctuations by the plastic motion of vortices, so that the system behaves as a noise correlation ratchet. The transverse ratchet effect is much more pronounced in the conformal arrays than in random gradient arrays and is absent in square gradient arrays due the different nature of the vortex flow in each geometry. We show that a series of reversals can occur in the transverse ratchet effect due to changes in the vortex flow across the pinning gradient as a function of vortex filling, pinning strength, and ac amplitude. We also consider the case where a dc drive applied perpendicular to the substrate asymmetry direction generates a net flow of vortices perpendicular to the dc drive, producing what is known as a geometric or drift ratchet that again arises due to non-Gaussian dynamically generated fluctuations. The drift ratchet is more efficient than the ac driven ratchet and also exhibits a series of reversals for varied parameters. Lastly, our results should be general to a wide class of systems undergoing nonequilibrium dynamics on conformal substrates, such as colloidal particles on optical traps.« less

Molecular mechanisms underlying deoxy‐ADP.Pi activation of pre‐powerstroke myosin

PubMed Central

Nowakowski, Sarah G.

2017-01-01

Abstract Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2‐deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross‐bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross‐bridge formation and reveal a potential mechanism that may underlie dATP‐induced improvements in cardiac function. PMID:28097776

Conformal coating of highly structured surfaces

DOEpatents

Ginley, David S.; Perkins, John; Berry, Joseph; Gennett, Thomas

2012-12-11

Method of applying a conformal coating to a highly structured substrate and devices made by the disclosed methods are disclosed. An example method includes the deposition of a substantially contiguous layer of a material upon a highly structured surface within a deposition process chamber. The highly structured surface may be associated with a substrate or another layer deposited on a substrate. The method includes depositing a material having an amorphous structure on the highly structured surface at a deposition pressure of equal to or less than about 3 mTorr. The method may also include removing a portion of the amorphous material deposited on selected surfaces and depositing additional amorphous material on the highly structured surface.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

PubMed Central

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

2015-01-01

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

Method and apparatus for forming conformal SiN.sub.x films

DOEpatents

Wang, Qi

2007-11-27

A silicon nitride film formation method includes: Heating a substrate to be subjected to film formation to a substrate temperature; heating a wire to a wire temperature; supplying silane, ammonia, and hydrogen gases to the heating member; and forming a silicon nitride film on the substrate.

Conformational dynamics of Peb4 exhibit "mother's arms" chain model: a molecular dynamics study.

PubMed

Dantu, Sarath Chandra; Khavnekar, Sagar; Kale, Avinash

2017-08-01

Peb4 from Campylobacter jejuni is an intertwined dimeric, periplasmic holdase, which also exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Peb4 gene deletion alters the outer membrane protein profile and impairs cellular adhesion and biofilm formation for C. jejuni. Earlier crystallographic study has proposed that the PPIase domains are flexible and might form a cradle for holding the substrate and these aspects of Peb4 were explored using sub-microsecond molecular dynamics simulations in solution environment. Our simulations have revealed that PPIase domains are highly flexible and undergo a large structural change where they move apart from each other by 8 nm starting at .5 nm. Further, this large conformational change renders Peb4 as a compact protein with crossed-over conformation, forms a central cavity, which can "cradle" the target substrate. As reported for other chaperone proteins, flexibility of linker region connecting the chaperone and PPIase domains is key to forming the "crossed-over" conformation. The conformational transition of the Peb4 protein from the X-ray structure to the crossed-over conformation follows the "mother's arms" chain model proposed for the FkpA chaperone protein. Our results offer insights into how Peb4 and similar chaperones can use the conformational heterogeneity at their disposal to perform its much-revered biological function.

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.

Molecular Dynamics Simulation of the Allosteric Regulation of eIF4A Protein from the Open to Closed State, Induced by ATP and RNA Substrates

PubMed Central

Meng, Hongqing; Li, Chaoqun; Wang, Yan; Chen, Guangju

2014-01-01

Background Eukaryotic initiation factor 4A (eIF4A) plays a key role in the process of protein translation initiation by facilitating the melting of the 5′ proximal secondary structure of eukaryotic mRNA for ribosomal subunit attachment. It was experimentally postulated that the closed conformation of the eIF4A protein bound by the ATP and RNA substrates is coupled to RNA duplex unwinding to promote protein translation initiation, rather than an open conformation in the absence of ATP and RNA substrates. However, the allosteric process of eIF4A from the open to closed state induced by the ATP and RNA substrates are not yet fully understood. Methodology In the present work, we constructed a series of diplex and ternary models of the eIF4A protein bound by the ATP and RNA substrates to carry out molecular dynamics simulations, free energy calculations and conformation analysis and explore the allosteric properties of eIF4A. Results The results showed that the eIF4A protein completes the conformational transition from the open to closed state via two allosteric processes of ATP binding followed by RNA and vice versa. Based on cooperative allosteric network analysis, the ATP binding to the eIF4A protein mainly caused the relative rotation of two domains, while the RNA binding caused the proximity of two domains via the migration of RNA bases in the presence of ATP. The cooperative binding of ATP and RNA for the eIF4A protein plays a key role in the allosteric transition. PMID:24465900

Structure of Human Pancreatic Lipase-Related Protein 2 with the Lid in an Open Conformation

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

Eydoux, Cecilia; Spinelli, Silvia; Davis, Tara L.

2008-10-02

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lidmore » is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.« less

Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation.

PubMed

Eydoux, Cécilia; Spinelli, Silvia; Davis, Tara L; Walker, John R; Seitova, Alma; Dhe-Paganon, Sirano; De Caro, Alain; Cambillau, Christian; Carrière, Frédéric

2008-09-09

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.

Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

PubMed Central

O’Mara, Megan L.; Mark, Alan E.

2014-01-01

ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg2+ with each NBD indicates that the coordination of ATP and Mg2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations. PMID:24632881

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

PubMed

Roy, Sourav; Karmakar, Tarak; Prahlada Rao, Vasudeva S; Nagappa, Lakshmeesha K; Balasubramanian, Sundaram; Balaram, Hemalatha

2015-05-01

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Polycrystalline Diamond Coating of Additively Manufactured Titanium for Biomedical Applications.

PubMed

Rifai, Aaqil; Tran, Nhiem; Lau, Desmond W; Elbourne, Aaron; Zhan, Hualin; Stacey, Alastair D; Mayes, Edwin L H; Sarker, Avik; Ivanova, Elena P; Crawford, Russell J; Tran, Phong A; Gibson, Brant C; Greentree, Andrew D; Pirogova, Elena; Fox, Kate

2018-03-14

Additive manufacturing using selective laser melted titanium (SLM-Ti) is used to create bespoke items across many diverse fields such as medicine, defense, and aerospace. Despite great progress in orthopedic implant applications, such as for "just in time" implants, significant challenges remain with regards to material osseointegration and the susceptibility to bacterial colonization on the implant. Here, we show that polycrystalline diamond coatings on these titanium samples can enhance biological scaffold interaction improving medical implant applicability. The highly conformable coating exhibited excellent bonding to the substrate. Relative to uncoated SLM-Ti, the diamond coated samples showed enhanced mammalian cell growth, enriched apatite deposition, and reduced microbial S. aureus activity. These results open new opportunities for novel coatings on SLM-Ti devices in general and especially show promise for improved biomedical implants.

Rapid prototyping of all-solution-processed multi-lengthscale electrodes using polymer-induced thin film wrinkling

PubMed Central

Gabardo, Christine M.; Adams-McGavin, Robert C.; Fung, Barnabas C.; Mahoney, Eric J.; Fang, Qiyin; Soleymani, Leyla

2017-01-01

Three-dimensional electrodes that are controllable over multiple lengthscales are very important for use in bioanalytical systems that integrate solid-phase devices with solution-phase samples. Here we present a fabrication method based on all-solution-processing and thin film wrinkling using smart polymers that is ideal for rapid prototyping of tunable three-dimensional electrodes and is extendable to large volume manufacturing. Although all-solution-processing is an attractive alternative to vapor-based techniques for low-cost manufacturing of electrodes, it often results in films suffering from low conductivity and poor substrate adhesion. These limitations are addressed here by using a smart polymer to create a conformal layer of overlapping wrinkles on the substrate to shorten the current path and embed the conductor onto the polymer layer. The structural evolution of these wrinkled electrodes, deposited by electroless deposition onto a nanoparticle seed layer, is studied at varying deposition times to understand its effects on structural parameters such as porosity, wrinkle wavelength and height. Furthermore, the effect of structural parameters on functional properties such as electro-active surface area and surface-enhanced Raman scattering is investigated. It is found that wrinkling of electroless-deposited thin films can be used to reduce sheet resistance, increase surface area, and enhance the surface-enhanced Raman scattering signal. PMID:28211898

Rapid prototyping of all-solution-processed multi-lengthscale electrodes using polymer-induced thin film wrinkling

NASA Astrophysics Data System (ADS)

Gabardo, Christine M.; Adams-McGavin, Robert C.; Fung, Barnabas C.; Mahoney, Eric J.; Fang, Qiyin; Soleymani, Leyla

2017-02-01

Three-dimensional electrodes that are controllable over multiple lengthscales are very important for use in bioanalytical systems that integrate solid-phase devices with solution-phase samples. Here we present a fabrication method based on all-solution-processing and thin film wrinkling using smart polymers that is ideal for rapid prototyping of tunable three-dimensional electrodes and is extendable to large volume manufacturing. Although all-solution-processing is an attractive alternative to vapor-based techniques for low-cost manufacturing of electrodes, it often results in films suffering from low conductivity and poor substrate adhesion. These limitations are addressed here by using a smart polymer to create a conformal layer of overlapping wrinkles on the substrate to shorten the current path and embed the conductor onto the polymer layer. The structural evolution of these wrinkled electrodes, deposited by electroless deposition onto a nanoparticle seed layer, is studied at varying deposition times to understand its effects on structural parameters such as porosity, wrinkle wavelength and height. Furthermore, the effect of structural parameters on functional properties such as electro-active surface area and surface-enhanced Raman scattering is investigated. It is found that wrinkling of electroless-deposited thin films can be used to reduce sheet resistance, increase surface area, and enhance the surface-enhanced Raman scattering signal.

Fabrication of fully transparent nanowire transistors for transparent and flexible electronics

NASA Astrophysics Data System (ADS)

Ju, Sanghyun; Facchetti, Antonio; Xuan, Yi; Liu, Jun; Ishikawa, Fumiaki; Ye, Peide; Zhou, Chongwu; Marks, Tobin J.; Janes, David B.

2007-06-01

The development of optically transparent and mechanically flexible electronic circuitry is an essential step in the effort to develop next-generation display technologies, including `see-through' and conformable products. Nanowire transistors (NWTs) are of particular interest for future display devices because of their high carrier mobilities compared with bulk or thin-film transistors made from the same materials, the prospect of processing at low temperatures compatible with plastic substrates, as well as their optical transparency and inherent mechanical flexibility. Here we report fully transparent In2O3 and ZnO NWTs fabricated on both glass and flexible plastic substrates, exhibiting high-performance n-type transistor characteristics with ~82% optical transparency. These NWTs should be attractive as pixel-switching and driving transistors in active-matrix organic light-emitting diode (AMOLED) displays. The transparency of the entire pixel area should significantly enhance aperture ratio efficiency in active-matrix arrays and thus substantially decrease power consumption.

Fabrication of fully transparent nanowire transistors for transparent and flexible electronics.

PubMed

Ju, Sanghyun; Facchetti, Antonio; Xuan, Yi; Liu, Jun; Ishikawa, Fumiaki; Ye, Peide; Zhou, Chongwu; Marks, Tobin J; Janes, David B

2007-06-01

The development of optically transparent and mechanically flexible electronic circuitry is an essential step in the effort to develop next-generation display technologies, including 'see-through' and conformable products. Nanowire transistors (NWTs) are of particular interest for future display devices because of their high carrier mobilities compared with bulk or thin-film transistors made from the same materials, the prospect of processing at low temperatures compatible with plastic substrates, as well as their optical transparency and inherent mechanical flexibility. Here we report fully transparent In(2)O(3) and ZnO NWTs fabricated on both glass and flexible plastic substrates, exhibiting high-performance n-type transistor characteristics with approximately 82% optical transparency. These NWTs should be attractive as pixel-switching and driving transistors in active-matrix organic light-emitting diode (AMOLED) displays. The transparency of the entire pixel area should significantly enhance aperture ratio efficiency in active-matrix arrays and thus substantially decrease power consumption.

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

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

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less

The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

DOE PAGES

Patel, Meha P.; Hu, Liya; Stojanoski, Vlatko; ...

2017-06-14

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzymemore » along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.« less

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

PubMed

Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

2016-08-01

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Monolayer and/or few-layer graphene on metal or metal-coated substrates

DOEpatents

Sutter, Peter Werner; Sutter, Eli Anguelova

2015-04-14

Disclosed is monolayer and/or few-layer graphene on metal or metal-coated substrates. Embodiments include graphene mirrors. In an example, a mirror includes a substrate that has a surface exhibiting a curvature operable to focus an incident beam onto a focal plane. A graphene layer conformally adheres to the substrate, and is operable to protect the substrate surface from degradation due to the incident beam and an ambient environment.

A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

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

Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla

2012-04-18

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change ofmore » a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.« less

Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters

NASA Astrophysics Data System (ADS)

Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng

2015-07-01

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Non-Amyloid Clients

PubMed Central

DeSantis, Morgan E.; Leung, Eunice H.; Sweeny, Elizabeth A.; Jackrel, Meredith E.; Cushman-Nick, Mimi; Neuhaus-Follini, Alexandra; Vashist, Shilpa; Sochor, Matthew A.; Knight, M. Noelle; Shorter, James

2012-01-01

Summary It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate non-co-operatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits co-operatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable ‘cross-β’ cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and non-amyloid clients, which impose distinct mechanical demands. PMID:23141537

The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

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

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.

2012-05-29

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR ofmore » arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.« less

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

Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters

PubMed Central

Cheng, Mary Hongying; Torres-Salazar, Delany; Gonzalez-Suarez, Aneysis D; Amara, Susan G; Bahar, Ivet

2017-01-01

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions. DOI: http://dx.doi.org/10.7554/eLife.25850.001 PMID:28569666

Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes

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

Jin, Lei; Wei, Wentao; Jiang, Yaobin

2009-11-04

SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into themore » conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.« less

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

PubMed

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

2015-04-17

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

DOE PAGES

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

2015-02-10

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

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

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

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

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

Heaslet, H.; Rosenfeld, R.; Giffin, M.

The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15 {angstrom} resolution are reported. In one crystal form HIV PR adopts an 'open' conformation with a 7.7 {angstrom} separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are 'curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3 {angstrom} resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. Themore » mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a 'curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the 'open' and 'curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire {beta}-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the {beta}-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors.« less

Structural mechanisms of chaperone mediated protein disaggregation

PubMed Central

Sousa, Rui

2014-01-01

The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID:25988153

Method and apparatus for enhancing vortex pinning by conformal crystal arrays

DOEpatents

Janko, Boldizsar; Reichhardt, Cynthia; Reichhardt, Charles; Ray, Dipanjan

2015-07-14

Disclosed is a method and apparatus for strongly enhancing vortex pinning by conformal crystal arrays. The conformal crystal array is constructed by a conformal transformation of a hexagonal lattice, producing a non-uniform structure with a gradient where the local six-fold coordination of the pinning sites is preserved, and with an arching effect. The conformal pinning arrays produce significantly enhanced vortex pinning over a much wider range of field than that found for other vortex pinning geometries with an equivalent number of vortex pinning sites, such as random, square, and triangular.

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

PubMed

Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

1989-12-08

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.

Replica exchange molecular dynamics simulations provide insight into substrate recognition by small heat shock proteins.

PubMed

Patel, Sunita; Vierling, Elizabeth; Tama, Florence

2014-06-17

The small heat shock proteins (sHSPs) are a virtually ubiquitous and diverse group of molecular chaperones that can bind and protect unfolding proteins from irreversible aggregation. It has been suggested that intrinsic disorder of the N-terminal arm (NTA) of sHSPs is important for substrate recognition. To investigate conformations of the NTA that could recognize substrates we performed replica exchange molecular dynamics simulations. Behavior at normal and stress temperatures of the dimeric building blocks of dodecameric HSPs from wheat (Ta16.9) and pea (Ps18.1) were compared because they display high sequence similarity, but Ps18.1 is more efficient in binding specific substrates. In our simulations, the NTAs of the dimer are flexible and dynamic; however, rather than exhibiting highly extended conformations they retain considerable α-helical character and contacts with the conserved α-crystallin domain (ACD). Network analysis and clustering methods reveal that there are two major conformational forms designated either "open" or "closed" based on the relative position of the two NTAs and their hydrophobic solvent accessible surface area. The equilibrium constant for the closed to open transition is significantly different for Ta16.9 and Ps18.1, with the latter showing more open conformations at elevated temperature correlated with its more effective chaperone activity. In addition, the Ps18.1 NTAs have more hydrophobic solvent accessible surface than those of Ta16.9. NTA hydrophobic patches are comparable in size to the area buried in many protein-protein interactions, which would enable sHSPs to bind early unfolding intermediates. Reduced interactions of the Ps18.1 NTAs with each other and with the ACD contribute to the differences in dynamics and hydrophobic surface area of the two sHSPs. These data support a major role for the conformational equilibrium of the NTA in substrate binding and indicate features of the NTA that contribute to sHSP chaperone efficiency. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

APE1 incision activity at abasic sites in tandem repeat sequences.

PubMed

Li, Mengxia; Völker, Jens; Breslauer, Kenneth J; Wilson, David M

2014-05-29

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains. Published by Elsevier Ltd.

Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

PubMed Central

Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

2016-01-01

The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

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.

Molecular Dynamics Simulations of the Human Glucose Transporter GLUT1

PubMed Central

Park, Min-Sun

2015-01-01

Glucose transporters (GLUTs) provide a pathway for glucose transport across membranes. Human GLUTs are implicated in devastating diseases such as heart disease, hyper- and hypo-glycemia, type 2 diabetes and caner. The human GLUT1 has been recently crystalized in the inward-facing open conformation. However, there is no other structural information for other conformations. The X-ray structures of E. coli Xylose permease (XylE), a glucose transporter homolog, are available in multiple conformations with and without the substrates D-xylose and D-glucose. XylE has high sequence homology to human GLUT1 and key residues in the sugar-binding pocket are conserved. Here we construct a homology model for human GLUT1 based on the available XylE crystal structure in the partially occluded outward-facing conformation. A long unbiased all atom molecular dynamics simulation starting from the model can capture a new fully opened outward-facing conformation. Our investigation of molecular interactions at the interface between the transmembrane (TM) domains and the intracellular helices (ICH) domain in the outward- and inward-facing conformation supports that the ICH domain likely stabilizes the outward-facing conformation in GLUT1. Furthermore, inducing a conformational transition, our simulations manifest a global asymmetric rocker switch motion and detailed molecular interactions between the substrate and residues through the water-filled selective pore along a pathway from the extracellular to the intracellular side. The results presented here are consistent with previously published biochemical, mutagenesis and functional studies. Together, this study shed light on the structure and functional relationships of GLUT1 in multiple conformational states. PMID:25919356

Conformational flexibility in the flap domains of ligand-free HIV protease.

PubMed

Heaslet, Holly; Rosenfeld, Robin; Giffin, Mike; Lin, Ying Chuan; Tam, Karen; Torbett, Bruce E; Elder, John H; McRee, Duncan E; Stout, C David

2007-08-01

The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15 A resolution are reported. In one crystal form HIV PR adopts an 'open' conformation with a 7.7 A separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are 'curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3 A resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. The mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a 'curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the 'open' and 'curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire beta-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the beta-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors.

Chain Conformation near the Buried Interface in Nanoparticle-Stabilized Polymer Thin Films

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

Barkley, Deborah A.; Jiang, Naisheng; Sen, Mani

It is known that when nanoparticles are added to polymer thin films, they often migrate to the film-substrate interface and form an “immobile interfacial layer”, which has been believed as the origin of suppression of dewetting. We here report an alternative mechanism of dewetting suppression from the structural aspect of a polymer. Dodecane thiol-functionalized gold (Au) nanoparticles embedded in PS thin films prepared on Si substrates were used as a model. It was found that thermal annealing promotes irreversible polymer adsorption onto the substrate surface along with the surface migration of the nanoparticles. We also revealed that the surface migrationmore » causes additional nanoconfined space for the adsorbed polymer chains. As a result, the self-organization process of the strongly adsorbed polymer chains on the solid surface was so hindered that the chain conformations were randomized and expanded in the film normal direction. Here, the resultant chain conformation allows the interpenetration between free chains and the adsorbed chains, promoting adhesion and hence stabilizing the thin film.« less

Chain Conformation near the Buried Interface in Nanoparticle-Stabilized Polymer Thin Films

DOE PAGES

Barkley, Deborah A.; Jiang, Naisheng; Sen, Mani; ...

2017-09-26

It is known that when nanoparticles are added to polymer thin films, they often migrate to the film-substrate interface and form an “immobile interfacial layer”, which has been believed as the origin of suppression of dewetting. We here report an alternative mechanism of dewetting suppression from the structural aspect of a polymer. Dodecane thiol-functionalized gold (Au) nanoparticles embedded in PS thin films prepared on Si substrates were used as a model. It was found that thermal annealing promotes irreversible polymer adsorption onto the substrate surface along with the surface migration of the nanoparticles. We also revealed that the surface migrationmore » causes additional nanoconfined space for the adsorbed polymer chains. As a result, the self-organization process of the strongly adsorbed polymer chains on the solid surface was so hindered that the chain conformations were randomized and expanded in the film normal direction. Here, the resultant chain conformation allows the interpenetration between free chains and the adsorbed chains, promoting adhesion and hence stabilizing the thin film.« less

SU-E-T-211: Comparison of Seven New TrueBeam Linacs with Enhanced Beam Data Conformance Using a Beam Comparison Software Tool

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

Grzetic, S; Hessler, J; Gupta, N

2015-06-15

Purpose: To develop an independent software tool to assist in commissioning linacs with enhanced beam conformance, as well as perform ongoing QA for dosimetrically equivalent linacs. Methods: Linac manufacturers offer enhanced beam conformance as an option to allow for clinics to complete commissioning efficiently, as well as implement dosimetrically equivalent linacs. The specification for enhanced conformance includes PDD as well as profiles within 80% FWHM. Recently, we commissioned seven Varian TrueBeam linacs with enhanced beam conformance. We developed a software tool in Visual Basic to allow us to load the reference beam data and compare our beam data during commissioningmore » to evaluate enhanced beam conformance. This tool also allowed us to upload our beam data used for commissioning our dosimetrically equivalent beam models to compare and tweak each of our linac beams to match our modelled data in Varian’s Eclipse TPS. This tool will also be used during annual QA of the linacs to compare our beam data to our baseline data, as required by TG-142. Results: Our software tool was used to check beam conformance for seven TrueBeam linacs that we commissioned in the past six months. Using our tool we found that the factory conformed linacs showed up to 3.82% difference in their beam profile data upon installation. Using our beam comparison tool, we were able to adjust the energy and profiles of our beams to accomplish a better than 1.00% point by point data conformance. Conclusion: The availability of quantitative comparison tools is essential to accept and commission linacs with enhanced beam conformance, as well as to beam match multiple linacs. We further intend to use the same tool to ensure our beam data conforms to the commissioning beam data during our annual QA in keeping with the requirements of TG-142.« 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

Pyrimidine homoribonucleosides: synthesis, solution conformation, and some biological properties.

PubMed

Lassota, P; Kuśmierek, J T; Stolarski, R; Shugar, D

1987-05-01

Conversion of uridine and cytidine to their 5'-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto unknown homouridine (1-(5'-deoxy-beta-D-allofuranosyl)uracil) and homocytidine (1-(5'-deoxy-beta-D-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5'-CH2OH chain is extended by one carbon to CH2CH2OH. Homocytidine was also phosphorylated to its 6'-phosphate and 6'-pyrophosphate analogues. In addition, it was converted, via its 2,2'-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1H and 13C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homocytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5'-nucleotidase, homoCDP was a potent inhibitor of this enzyme (Ki approximately 6 microM). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.

Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change*s

PubMed Central

Li, Yi-Ching; Chiang, Chia-Wang; Yeh, Hui-Chun; Hsu, Pei-Yung; Whitby, Frank G.; Wang, Lee-Ho; Chan, Nei-Li

2008-01-01

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H2. PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H2, leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels. PMID:18032380

Protein adsorption on tailored substrates: long-range forces and conformational changes

NASA Astrophysics Data System (ADS)

Bellion, M.; Santen, L.; Mantz, H.; Hähl, H.; Quinn, A.; Nagel, A.; Gilow, C.; Weitenberg, C.; Schmitt, Y.; Jacobs, K.

2008-10-01

Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, α-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

Tensile cracking of a brittle conformal coating on a rough substrate

DOE PAGES

Reedy, Jr., E. D.

2016-04-07

This note examines the effect of interfacial roughness on the initiation and growth of channel cracks in a brittle film. A conformal film with cusp-like surface flaws that replicate the substrate roughness is investigated. This type of surface flaw is relatively severe in the sense that stress diverges as the cusp-tip is approached (i.e., there is a power-law stress singularity). For the geometry and range of film properties considered, the analysis suggests that smoothing the substrate could substantially increase the film’s resistance to the formation of the through-the-thickness cracks that precede channel cracking. Furthermore, smoothing the substrate’s surface has amore » relatively modest effect on the film stress needed to propagate a channel crack.« less

Controlled growth of vertically aligned carbon nanotubes on metal substrates

NASA Astrophysics Data System (ADS)

Gao, Zhaoli

Carbon nanotube (CNT) is a fascinating material with extraordinary electrical thermal and mechanical properties. Growing vertically aligned CNT (VACNT) arrays on metal substrates is an important step in bringing CNT into practical applications such as thermal interface materials (TIMs) and microelectrodes. However, the growth process is challenging due to the difficulties in preventing catalyst diffusion and controlling catalyst dewetting on metal substrates with physical surface heterogeneity. In this work, the catalyst diffusion mechanism and catalyst dewetting theory were studied for the controlled growth of VACNTs on metal substrates. The diffusion time of the catalyst, the diffusion coefficients for the catalyst in the substrate materials and the number density of catalyst nanoparticles after dewetting are identified as the key parameters, based on which three strategies are developed. Firstly, a fast-heating catalyst pretreatment strategy was used, aiming at preserving the amount of catalyst prior to CNT growth by reducing the catalyst diffusion time. The catalyst lifetime is extended from half an hour to one hour on a patterned Al thin film and a VACNT height of 106 mum, about twenty fold of that reported in the literature, was attained. Secondly, a diffusion barrier layer strategy is employed for a reduction of catalyst diffusion into the substrate materials. Enhancement of VACNT growth on Cu substrates was achieved by adopting a conformal Al2O 3 diffusion barrier layer fabricated by a specially designed atomic layer deposition (ALD) system. Lastly, a novel catalyst glancing angle deposition (GLAD) strategy is performed to manipulate the morphology of a relatively thick catalyst on metal substrates with physical surface heterogeneity, aiming to obtain uniform and dense catalyst nanoparticles after dewetting in the pretreatment process for enhanced VACNT growth. We are able to control the VACNT growth conditions on metal substrates in terms of their distribution, heights and alignments. Catalyst loss is controlled by the catalyst diffusion time and catalyst diffusion coefficients. A shorter catalyst diffusion time and smaller diffusion coefficient enhance VACNT growth on metals due to reduced catalyst loss during the pretreatment process. The dewetting behaviors of the thin film catalysts are influenced by the physical surface heterogeneity of the substrates which leads to non-uniform growth of VACNTs. The GLAD process facilitates the deposition of a relatively thick catalyst layer for the creation of dense and uniform catalyst nanoparticles. Applications of VACNT-metal structures in TIMs and microelectrodes are demonstrated. The VACNT-TIMs fabricated on Al alloy substrates have a typical thermal contact resistivity of 17.1 mm2˙K/W and their effective application in high-brightness LED thermal management was demonstrated. Electrochemical characterization was carried out on VACNT microelectrodes for the development of high resolution retinal prostheses and a satisfactory electrochemical property was again demonstrated.

Method for ultra-fast boriding

DOEpatents

Erdemir, Ali; Sista, Vivekanand; Kahvecioglu, Ozgenur; Eryilmaz, Osman Levent

2017-01-31

An article of manufacture and method of forming a borided material. An electrochemical cell is used to process a substrate to deposit a plurality of borided layers on the substrate. The plurality of layers are co-deposited such that a refractory metal boride layer is disposed on a substrate and a rare earth metal boride conforming layer is disposed on the refractory metal boride layer.

Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles

PubMed Central

Rosenzweig, Rina; Sekhar, Ashok; Nagesh, Jayashree; Kay, Lewis E

2017-01-01

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state. DOI: http://dx.doi.org/10.7554/eLife.28030.001 PMID:28708484

Lys98 Substitution in Human AP Endonuclease 1 Affects the Kinetic Mechanism of Enzyme Action in Base Excision and Nucleotide Incision Repair Pathways

PubMed Central

Timofeyeva, Nadezhda A.; Koval, Vladimir V.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Fedorova, Olga S.

2011-01-01

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5′-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates. PMID:21912662

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

Jacob, Reed B.; Ding, Feng; Ye, Dongmei

Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allostericallymore » coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (k cat =16.63 s -1), such as T199I/T54I (899.5 s -1) and C227V/T199I/T54I (848 s -1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.« less

Molecular modeling studies of substrate binding by penicillin acylase.

PubMed

Chilov, G G; Stroganov, O V; Svedas, V K

2008-01-01

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

Conformational and chemical selection by a trans-acting editing domain

PubMed Central

Danhart, Eric M.; Bakhtina, Marina; Cantara, William A.; Kuzmishin, Alexandra B.; Ma, Xiao; Sanford, Brianne L.; Vargas-Rodriguez, Oscar; Košutić, Marija; Goto, Yuki; Suga, Hiroaki; Nakanishi, Kotaro; Micura, Ronald; Musier-Forsyth, Karin

2017-01-01

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that edits Ala-tRNAPro, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a nonhydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation. Backbone 15N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups. PMID:28768811

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.

Modeling of a C-end rule peptide adsorbed onto gold nanoparticles.

PubMed

Triguero, Jordi; Flores-Ortega, Alejandra; Zanuy, David; Alemán, Carlos

2018-01-01

The RPAR peptide, a prototype C-end Rule (CendR) sequence that binds to neuropilin-1 (NRP-1), has potential therapeutic uses as internalization trigger in anticancer nanodevices. Recently, the functionalization of gold nanoparticles with CendR peptides has been proved to be a successful strategy to target the NRP-1 receptor in prostate cancer cells. In this work, we investigate the influence of two gold surface facets, (100) and (111), on the conformational preferences of RPAR using molecular dynamics simulations. Both clustering and conformational analyses revealed that the peptide backbone becomes very rigid upon adsorption onto gold, which is a very fast and favored process, the only flexibility being attributed to the side chains of the two Arg residues. Thus, the different components of RPAR tend to adopt an elongated shape, which is characterized by the pseudo-extended conformation of both the backbone and the Arg side chains. This conformation is very different from the already known bioactive conformation, indicating that RPAR is drastically affected by the substrate. Interestingly, the preferred conformations of the peptide adsorbed onto gold facets are not stabilized by salt bridges and/or specific intramolecular hydrogen bonds, which represent an important difference with respect to the conformations found in other environments (e.g. the peptide in solution and interacting with NRP-1 receptor). However, the conformational changes induced by the substrate are not detrimental for the use of gold nanoparticles as appropriate vehicles for the transport and targeted delivery of the RPAR. Thus, once their high affinity for the NRP-1 receptor induces the targeted delivery of the elongated peptide molecules from the gold nanoparticles, the lack of intramolecular interactions facilitates their evolution towards the bioactive conformation, increasing the therapeutic efficacy of the peptide. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Arginine Kinase. Joint Crystallographic & NMR RDC Analyses link Substrate-Associated Motions to Intrinsic Flexibility

PubMed Central

Niu, Xiaogang; Brüschweiler-Li, Lei; Davulcu, Omar; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

2010-01-01

The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a “high energy” phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and –bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection. PMID:21075117

Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction.

PubMed

Zhang, Xue-Jian; Wang, Xiao-Wei; Sun, Jiaxing; Su, Chao; Yang, Shuguang; Zhang, Wen-Bin

2018-05-16

Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to bio-signals (Ca2+, TFP, NADPH, etc.) and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multi-layer set-up exhibits a synergistic enhancement of DHFR activity with increasing number of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.

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

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT.

PubMed

Merkle, Patrick S; Gotfryd, Kamil; Cuendet, Michel A; Leth-Espensen, Katrine Z; Gether, Ulrik; Loland, Claus J; Rand, Kasper D

2018-05-01

LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na + - and K + -dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drives LeuT from a substrate-bound, outward-facing occluded conformation toward an inward-facing open state. These hitherto unknown, large-scale conformational changes in functionally important transmembrane segments, observed for LeuT in detergent-solubilized form and when embedded in a native-like phospholipid bilayer, could be of physiological relevance for the translocation process.

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

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Electron crystallography reveals that substrate release from the PTS IIC glucose transporter is coupled to a subtle conformational change.

PubMed

Kalbermatter, David; Chiu, Po-Lin; Jeckelmann, Jean-Marc; Ucurum, Zöhre; Walz, Thomas; Fotiadis, Dimitrios

2017-07-01

The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is a structurally and functionally complex system that mediates sugar uptake in bacteria. Besides several soluble subunits, the glucose-specific PTS includes the integral membrane protein IICB that couples the transmembrane transport of glucose to its phosphorylation. Here, we used electron crystallography of sugar-embedded tubular crystals of the glucose-specific IIC transport domain from Escherichia coli (ecIIC glc ) to visualize the structure of the transporter in the presence and absence of its substrate. Using an in vivo transport assay and binding competition experiments, we first established that, while it transports d-glucose, ecIIC glc does not bind l-glucose. We then determined the projection structure of ecIIC glc from tubular crystals embedded in d- and l-glucose and found a subtle conformational change. From comparison of the ecIIC glc projection maps with crystal structures of other IIC transporters, we can deduce that the transporter adopts an inward-facing conformation, and that the maps in the presence and absence of the substrate reflect the transporter before and after release of the transported glucose into the cytoplasm. The transition associated with substrate release appears to require a subtle structural rearrangement in the region that includes hairpin 1. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Studies of E. coli Topoisomerase III-DNA Complexes Reveal A Novel Type IA Topoisomerase-DNA Conformational Intermediate

PubMed Central

Changela, Anita; DiGate, Russell J.; Mondragón, Alfonso

2007-01-01

Summary E. coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5′ phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an 8-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding. PMID:17331537

Role of a GAG Hinge in the Nucleotide-induced Conformational Change Governing Nucleotide Specificity by T7 DNA Polymerase*

PubMed Central

Jin, Zhinan; Johnson, Kenneth A.

2011-01-01

A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. PMID:20978284

Conformable actively multiplexed high-density surface electrode array for brain interfacing

DOEpatents

Rogers, John; Kim, Dae-Hyeong; Litt, Brian; Viventi, Jonathan

2015-01-13

Provided are methods and devices for interfacing with brain tissue, specifically for monitoring and/or actuation of spatio-temporal electrical waveforms. The device is conformable having a high electrode density and high spatial and temporal resolution. A conformable substrate supports a conformable electronic circuit and a barrier layer. Electrodes are positioned to provide electrical contact with a brain tissue. A controller monitors or actuates the electrodes, thereby interfacing with the brain tissue. In an aspect, methods are provided to monitor or actuate spatio-temporal electrical waveform over large brain surface areas by any of the devices disclosed herein.

Strain-Engineering of Giant Pseudo-Magnetic Fields in Graphene/Boron Nitride (BN) Periodic Nanostructures

NASA Astrophysics Data System (ADS)

Hsu, Chen-Chih; Wang, Jiaqing; Teague, Marcus; Chen, Chien-Chang; Yeh, Nai-Chang

2015-03-01

Ideal graphene is strain-free whereas non-trivial strain can induce pseudo-magnetic fields as predicted theoretically and manifested experimentally. Here we employ nearly strain-free single-domain graphene, grown by plasma-enhanced chemical vapor deposition (PECVD) at low temperatures, to induce controlled strain by placing the PECVD-graphene on substrates containing engineered nanostructures. We fabricate periodic pyramid nanostructures (typically 100 ~ 200 nm laterally and 10 ~ 60 nm in height) on Si substrates by focused ion beam, and determine the topography of these nanostructures using atomic force microscopy and scanning electron microscopy after we transferred monolayer h-BN followed by PECVD-graphene onto these substrates. We find both layers conform well to the nanostructures so that we can control the size, arrangement, separation, and shape of the nanostructures to generate desirable pseudo-magnetic fields. We also employ molecular dynamics simulation to determine the displacement of carbon atoms under a given nanostructure. The pseudo-magnetic field thus obtained is ~150T in the center, relatively homogeneous over 50% of the area, and drops off precipitously near the edge. These findings are extended to arrays of nanostructures and compared with topographic and spectroscopic studies by STM. Supported by NSF.

Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

PubMed

Yap, F L; Zhang, Y

2007-01-15

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

Simultaneous use of in silico design and a correlated mutation network as a tool to efficiently guide enzyme engineering.

PubMed

Nobili, Alberto; Tao, Yifeng; Pavlidis, Ioannis V; van den Bergh, Tom; Joosten, Henk-Jan; Tan, Tianwei; Bornscheuer, Uwe T

2015-03-23

In order to improve the efficiency of directed evolution experiments, in silico multiple-substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3-phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta-analysis of the docking conformations in wild-type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15-fold improved enantioselectivity was found. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity.

PubMed

Szeltner, Z; Alshafee, I; Juhász, T; Parvari, R; Polgár, L

2005-10-01

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.

Compressive Properties of Open-Cell Al Hybrid Foams at Different Temperatures

PubMed Central

Liu, Jiaan; Si, Fujian; Zhu, Xianyong; Liu, Yaohui; Zhang, Jiawei; Liu, Yan; Zhang, Chengchun

2017-01-01

Hybrid Ni/Al foams were fabricated by depositing electroless Ni–P (EN) coatings on open-cell Al foam substrate to obtain enhanced mechanical properties. The microstructure, chemical components and phases of the hybrid foams were observed and analyzed by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD), respectively. The mechanical properties of the foams were studied by compressive tests at different temperatures. The experiment results show that the coating is mainly composed of Ni and P elements. There was neither defect at the interface nor crack in the coatings, indicating that the EN coatings had fine adhesion to the Al substrate. The compressive strengths and energy absorption capacities of the as-received foam and hybrid foams decrease with the increasing testing temperatures, but the hybrid foams exhibit a lower decrement rate than the as-received foam. This might be attributed to the different failure mechanisms at different testing temperatures, which is conformed by fractography observation. PMID:28772456

Tailoring plasmonic nanoparticles and fractal patterns

NASA Astrophysics Data System (ADS)

Rosa, Lorenzo; Juodkazis, Saulius

2011-12-01

We studied new three-dimensional tailoring of nano-particles by ion-beam and electron-beam lithographies, aiming for features and nano-gaps down to 10 nm size. Electron-beam patterning is demonstrated for 2D fabrication in combination with plasmonic metal deposition and lift-off, with full control of spectral features of plasmonic nano-particles and patterns on dielectric substrates. We present wide-angle bow-tie rounded nano-antennas whose plasmonic resonances achieve strong field enhancement at engineered wavelength range, and show how the addition of fractal patterns defined by standard electron beam lithography achieve light field enhancement from visible to far-IR spectral range and scalable up towards THz band. Field enhancement is evaluated by FDTD modeling on full-3D simulation domains using complex material models, showing the modeling method capabilities and the effect of staircase approximations on field enhancement and resonance conditions, especially at metal corners, where a minimum rounding radius of 2 nm is resolved and a five-fold reduction of spurious ringing at sharp corners is obtained by the use of conformal meshing.

Conformational Changes and Substrate Recognition in Pseudomonas aeruginosa d-Arginine Dehydrogenase

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

Fu, Guoxing; Yuan, Hongling; Li, Congran

2010-11-15

DADH catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding {alpha}-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 {angstrom} resolution and its complexes with iminoarginine (DADH{sub red}/iminoarginine) and iminohistidine (DADH{sub red}/iminohistidine) at 1.30 {angstrom} resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADH{sub red}/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an 'active site lid',more » similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in k{sub cat}/K{sub m} values for D-arginine and D-histidine. Comparison of the kinetic data for the activity of DADH on different D-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.« less

Crystal structures reveal an induced-fit binding of a substrate-like Aza-peptide epoxide to SARS coronavirus main peptidase.

PubMed

Lee, Ting-Wai; Cherney, Maia M; Liu, Jie; James, Karen Ellis; Powers, James C; Eltis, Lindsay D; James, Michael N G

2007-02-23

The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.

The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange

PubMed Central

Clerici, Marcello; Luna-Vargas, Mark P. A.; Faesen, Alex C.; Sixma, Titia K.

2014-01-01

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP–Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP–Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP–Ubl domain promotes a change of a switching loop near the active site. This ‘allosteric regulation of product discharge’ provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes. PMID:25404403

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

Changela, Anita; DiGate, Russell J.; Mondragon, Alfonso

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5{prime} phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is boundmore » along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.« less

Substrate specificity in enzymatic fluorination. The fluorinase from Streptomyces cattleya accepts 2′-deoxyadenosine substrates†

PubMed Central

Cobb, Steven L.; Deng, Hai; McEwan, Andrew R.; Naismith, James H.; O’Hagan, David; Robinson, David A.

2012-01-01

The fluorinase enzyme from Streptomyces cattleya displays an unusual ability in biocatalysis in that it forms a C–F bond. We now report that the enzyme will accept 2′-deoxyadenosine in place of adenosine substrates, and structural evidence reveals a reorganisation in hydrogen bonding to accommodate this substrate series. It emerges from this study that the enzyme does not require a planar ribose conformation of the substrate to catalyse C–F bond formation. PMID:16604208

Investigation of an "alternate water supply system" in enzymatic hydrolysis in the processive endocellulase Cel7A from Rasamsonia emersonii by molecular dynamics simulation.

PubMed

Sun, Xun; Qian, Meng-Dan; Guan, Shan-Shan; Shan, Ya-Ming; Dong, Ying; Zhang, Hao; Wang, Song; Han, Wei-Wei

2017-02-01

Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross-correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water-supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β-sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond. © 2016 Wiley Periodicals, Inc.

Fabrication of trough-shaped solar collectors

DOEpatents

Schertz, William W.

1978-01-01

There is provided a radiant energy concentration and collection device formed of a one-piece thin-walled plastic substrate including a plurality of nonimaging troughs with certain metallized surfaces of the substrate serving as reflective side walls for each trough. The one-piece plastic substrate is provided with a seating surface at the bottom of each trough which conforms to the shape of an energy receiver to be seated therein.

Syk Inhibits the Activity of Protein Kinase A by Phosphorylating Tyrosine 330 of the Catalytic Subunit*

PubMed Central

Yu, Shuai; Huang, He; Iliuk, Anton; Wang, Wen-Horng; Jayasundera, Keerthi B.; Tao, W. Andy; Post, Carol B.; Geahlen, Robert L.

2013-01-01

The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified PKA as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on Tyr-330. Tyr-330 lies within the adenosine-binding motif in the C-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Tyr-330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide-binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Tyr-330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, cAMP-responsive element-binding protein (CREB), is inhibited in cells expressing Syk but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk, and its phosphorylation on Tyr-330 inhibits its participation in downstream signaling pathways. PMID:23447535

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.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

PubMed

Lopes, Jose L S; Beltramini, Leila M; Wallace, Bonnie A; Araujo, Ana P U

2015-01-01

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

PubMed Central

Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

2013-01-01

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

Coarse-grained Simulations of Substrate Export through Multidrug Efflux Transporter AcrB

NASA Astrophysics Data System (ADS)

Jewel, Yead; Dutta, Prashanta; Liu, Jin

2017-11-01

The treatment of bacterial infectious diseases hampered by the overexpression of multidrug resistance (MDR) systems. The MDR system actively pumps the antibiotic drugs as well as other toxic compounds out of the cells. During the pumping, AcrB (one of the key MDR components) undergoes a series of large-scale proton/substrate dependent conformational changes. In this work, we implement a hybrid coarse-grained PACE force field that couples the united-atom protein model with the coarse-grained MARTINI water/lipid, to investigate the conformational changes of AcrB. We first develop the substrate force field which is compatible with PACE, then we implement the force field to explore large scale structural changes of AcrB in microsecond simulations. The effects of the substrate and the protonation states of two key residues: Asp407 and Asp408, are investigated. Our results show that the drug export through AcrB is proton as well as substrate dependent. Our simulations explain molecular mechanisms of substrate transport through AcrB complex, as well as provide valuable insights for designing proper antibiotic drugs. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM122081.

Conformal Electroless Nickel Plating on Silicon Wafers, Convex and Concave Pyramids, and Ultralong Nanowires.

PubMed

Gill, Thomas Mark; Zhao, Jiheng; Berenschot, Erwin J W; Tas, Niels; Zheng, Xiaolin

2018-06-25

Nickel (Ni) plating has garnered great commercial interest, as it provides excellent hardness, corrosion resistance, and electrical conductivity. Though Ni plating on conducting substrates is commonly employed via electrodeposition, plating on semiconductors and insulators often necessitates electroless approaches. Corresponding plating theory for deposition on planar substrates was developed as early as 1946, but for substrates with micro- and nanoscale features, very little is known of the relationships between plating conditions, Ni deposition quality, and substrate morphology. Herein, we describe the general theory and mechanisms of electroless Ni deposition on semiconducting silicon (Si) substrates, detailing plating bath failures and establishing relationships between critical plating bath parameters and the deposited Ni film quality. Through this theory, we develop two different plating recipes: galvanic displacement (GD) and autocatalytic deposition (ACD). Neither recipe requires pretreatment of the Si substrate, and both methods are capable of depositing uniform Ni films on planar Si substrates and convex Si pyramids. In comparison, ACD has better tunability than GD, and it provides a more conformal Ni coating on complex and high-aspect-ratio Si structures, such as inverse fractal Si pyramids and ultralong Si nanowires. Our methodology and theoretical analyses can be leveraged to develop electroless plating processes for other metals and metal alloys and to generally provide direction for the adaptation of electroless deposition to modern applications.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change.

PubMed

Mandal, Soumit Sankar; Merz, Dale R; Buchsteiner, Maximilian; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

2017-06-06

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

PubMed Central

Mandal, Soumit Sankar; Buchsteiner, Maximilian; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

2017-01-01

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements. PMID:28533394

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

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

Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp

We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

DNA origami based Au–Ag-core–shell nanoparticle dimers with single-molecule SERS sensitivity† †Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA bands, SEM images, additional AFM images, FDTD simulations, additional reference spectra for Cy3 and detailed description of EF estimation, simulated absorption and scattering spectra. See DOI: 10.1039/c5nr08674d Click here for additional data file.

PubMed Central

Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.

2016-01-01

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. PMID:26892770

Structures of Substrate-And Inhibitor-Bound Adenosine Deaminase From a Human Malaria Parasite Show a Dramatic Conformational Change And Shed Light on Drug Selectivity

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

Larson, E.T.; Deng, W.; Krumm, B.E.

Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore, interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial ADA accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5{prime}-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificitymore » between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax ADA in complex with adenosine, guanosine, and the picomolar inhibitor 2{prime}-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes.« less

Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

PubMed

Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

2014-12-19

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy*

PubMed Central

Rout, Ashok K.; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

2014-01-01

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. PMID:25342746

Basic Residues R260 and K357 Affect the Conformational Dynamics of the Major Facilitator Superfamily Multidrug Transporter LmrP

PubMed Central

Wang, Wei; van Veen, Hendrik W.

2012-01-01

Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H+(Na+) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport. PMID:22761697

A diffuse reflectance comparative study of benzil inclusion within microcrystalline cellulose and beta-cyclodextrin.

PubMed

Vieira Ferreira, Luis F; Ferreira Machado, Isabel; Da Silva, José P; Oliveira, Anabela S

2004-02-01

Diffuse reflectance and laser-induced techniques were used to study photochemical and photophysical processes of benzil adsorbed on two solid powdered supports, microcrystalline cellulose and [small beta]-cyclodextrin. In both substrates, a distribution of ground-state benzil conformers exists, largely dominated by skew conformations where the carbonyl groups are twisted one to the other. Room temperature phosphorescence was observed in air-equilibrated samples in both cases. The decay times vary greatly and the largest lifetime was obtained for benzil/[small beta]-cyclodextrin, showing that this host's cavity accommodates benzil well, enhancing its room temperature phosphorescence. Triplet-triplet absorption of benzil entrapped in cellulose was detected and benzil ketyl radical formation also occurred. With benzil included into [small beta]-cyclodextrin, and following laser excitation, benzoyl radicals were detected on the millisecond timescale. Product analysis and identification of laser-irradiated benzil samples in the two hosts clearly showed that the main degradation photoproducts were benzoic acid and benzaldehyde. The main differences were a larger benzoic acid/benzaldehyde ratio in the case of cellulose and the formation of benzyl alcohol in this support.

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

PubMed

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2018-04-01

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog*

PubMed Central

Mulligan, Christopher; Mindell, Joseph A.

2013-01-01

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na+-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, GltPh, suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3–4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, GltPh is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved GltPh retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process. PMID:24155238

Label-free liquid crystal biosensor based on specific oligonucleotide probes for heavy metal ions.

PubMed

Yang, Shengyuan; Wu, Chao; Tan, Hui; Wu, Yan; Liao, Shuzhen; Wu, Zhaoyang; Shen, Guoli; Yu, Ruqin

2013-01-02

In this study, to enhance the capability of metal ions disturbing the orientation of liquid crystals (LCs), we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg(2+) ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg(2+), the specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N,N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. The optical signal of LC sensor has a visible change at the Hg(2+) concentration of low to 0.1 nM, showing good detection sensitivity. The cost-effective LC sensing method can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors.

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.

Synthesis and evaluation of conformationally restricted inhibitors of aspartate semialdehyde dehydrogenase.

PubMed

Evitt, Andrew S; Cox, Russell J

2011-05-01

Inhibitors of the enzyme aspartate semialdehyde dehydrogenase, a key biological target for the generation of a new class of antibiotic compounds, have been developed. To investigate improvements to binding within an inhibitor series, the lowering of the entropic barrier to binding through conformational restriction was investigated. A library of linear and cyclic substrate analogues was generated and computational docking used to aid in structure selection. The cyclic phosphonate inhibitor 18 was thus identified as complimentary to the enzyme active-site. Synthesis and in vitro inhibition assay revealed a K(i) of 3.8 mM against natural substrate, where the linear analogue of 18, compound 15, had previously shown no inhibitory activity. Two further inhibitors, phosphate analogue diastereoisomers 17a and 17b, were synthesised and also found to have low millimolar K(i) values. As a result of the computational docking investigations, a novel substrate binding interaction was discovered: hydrogen bonding between the substrate (phosphate hydroxy-group as the hydrogen bond donor) and the NADPH cofactor (2'-oxygen as the hydrogen bond acceptor).

Soft lubrication

NASA Astrophysics Data System (ADS)

Skotheim, Jan; Mahadevan, Laksminarayanan

2004-11-01

We study the lubrication of fluid-immersed soft interfaces and show that elastic deformation couples tangential and normal forces and thus generates lift. We consider materials that deform easily, due to either geometry (e.g a shell) or constitutive properties (e.g. a gel or a rubber), so that the effects of pressure and temperature on the fluid properties may be neglected. Four different system geometries are considered: a rigid cylinder moving tangentially to a soft layer coating a rigid substrate; a soft cylinder moving tangentially to a rigid substrate; a cylindrical shell moving tangentially to a rigid substrate; and finally a journal bearing coated with a thin soft layer, which being a conforming contact allows us to gauge the influence of contact geometry. In addition, for the particular case of a soft layer coating a rigid substrate we consider both elastic and poroelastic material responses. Finally, we consider the role of contact geometry in the context of the journal bearing, a conforming contact. For all these cases we find the same generic behavior: there is an optimal combination of geometric and material parameters that maximizes the dimensionless normal force as a function of the softness.

Flexible and conformable broadband metamaterial absorber with wide-angle and polarization stability for radar application

NASA Astrophysics Data System (ADS)

Chen, Huijie; Yang, Xiaoqing; Wu, Shiyue; Zhang, Di; Xiao, Hui; Huang, Kama; Zhu, Zhanxia; Yuan, Jianping

2018-01-01

In this work, a type of flexible, broadband electromagnetic microwave absorber is designed, fabricated and experimentally characterized. The absorber is composed of lumped resistors loaded frequency selective surface which is mounted on flexible substrate using silicone rubber and in turn backed by copper film. The simulated results show that an effective absorption (over 90%) bandwidth spans from 7.6 to 18.3 GHz, which covers both X (8-12 GHz) and Ku (12-18 GHz) bands, namely a 82.6% fraction bandwidth. And the bandwidth performs a good absorption response by varying the incident angle up to 60° for both TE and TM polarization. Moreover, the flexibility of the substrate enables the absorber conformably to bend and attach to cylinders of various radius without breakdown of the absorber. The designed structure has been fabricated and measured for both planar and conformable cases, and absorption responses show a good agreement of the broadband absorption feature with the simulated ones. This work has demonstrated specifically that proposed structure provides polarization-insensitive, wide-angle, flexible and conformable wideband absorption, which extends the absorber’s application to practical radar cross section reductions for radars and warships.

Flexible Near-Field Nanopatterning with Ultrathin, Conformal Phase Masks on Nonplanar Substrates for Biomimetic Hierarchical Photonic Structures.

PubMed

Kwon, Young Woo; Park, Junyong; Kim, Taehoon; Kang, Seok Hee; Kim, Hyowook; Shin, Jonghwa; Jeon, Seokwoo; Hong, Suck Won

2016-04-26

Multilevel hierarchical platforms that combine nano- and microstructures have been intensively explored to mimic superior properties found in nature. However, unless directly replicated from biological samples, desirable multiscale structures have been challenging to efficiently produce to date. Departing from conventional wafer-based technology, new and efficient techniques suitable for fabricating bioinspired structures are highly desired to produce three-dimensional architectures even on nonplanar substrates. Here, we report a facile approach to realize functional nanostructures on uneven microstructured platforms via scalable optical fabrication techniques. The ultrathin form (∼3 μm) of a phase grating composed of poly(vinyl alcohol) makes the material physically flexible and enables full-conformal contact with rough surfaces. The near-field optical effect can be identically generated on highly curved surfaces as a result of superior conformality. Densely packed nanodots with submicron periodicity are uniformly formed on microlens arrays with a radius of curvature that is as low as ∼28 μm. Increasing the size of the gratings causes the production area to be successfully expanded by up to 16 in(2). The "nano-on-micro" structures mimicking real compound eyes are transferred to flexible and stretchable substrates by sequential imprinting, facilitating multifunctional optical films applicable to antireflective diffusers for large-area sheet-illumination displays.

Catalysis by orotidine 5'-monophosphate decarboxylase: effect of 5-fluoro and 4'-substituents on the decarboxylation of two-part substrates.

PubMed

Goryanova, Bogdana; Spong, Krisztina; Amyes, Tina L; Richard, John P

2013-01-22

The syntheses of two novel truncated analogs of the natural substrate orotidine 5'-monophosphate (OMP) for orotidine 5'-monophosphate decarboxylase (OMPDC) with enhanced reactivity toward decarboxylation are reported: 1-(β-d-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5'-deoxy-5-fluoroorotidine (5'-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M⁻¹ s⁻¹) and 1-(β-d-erythrofuranosyl)orotic acid (EO, 0.026 M⁻¹ s⁻¹) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO₃²⁻), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in the rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E•FEO•HPO₃²⁻ complex for the reaction of FEO. The 4'-CH₃ and 4'-CH₂OH groups of 5'-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4'-CH₃ group of 5'-dFO and the 4'-CH₂OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4'-CH₃ substituent at 5'-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO.

Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel.

PubMed

Gencic, Simonida; Kelly, Kayla; Ghebreamlak, Selamawit; Duin, Evert C; Grahame, David A

2013-03-12

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO.

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.

Improved light output of plastic scintillator by a modified self-assembled photonic crystal

NASA Astrophysics Data System (ADS)

Chen, Liang; Zhu, Zhichao; Liu, Bo; Cheng, Chuanwei; Liu, Jinliang; Ruan, Jinlu; Zhang, Zhongbin; Ouyang, Xiaoping; Gu, Mu; Chen, Hong

2017-11-01

In this investigation, we have demonstrated that a modified self-assembled photonic crystal with conformal high refractive index material TiO2 can achieve a great enhancement of light extraction efficiency. A 2.26 fold wavelength- and angle-integrated enhancement ratio can be achieved. The conformal layer increases the number of leaky modes and thus improve the extraction efficiency. The enhancement is attributed to the leaky modes based on the individual microspheres with conformal layer. Their low quality factors with a broadband characteristic are advantageous to the broadband enhancement for the emission spectra of plastic scintillator. Furthermore, the dense conformal layers have excellent combination with the self-assembled microspheres and the whole preparation process cannot destroy the plastic scintillator, which is beneficial to the practical application.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

NASA Astrophysics Data System (ADS)

Venditti, Vincenzo; Tugarinov, Vitali; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius

2015-01-01

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Multiple Drug Transport Pathways through human P-Glycoprotein(†)

PubMed Central

McCormick, James W.; Vogel, Pia D.; Wise, John G.

2015-01-01

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

Multiple Drug Transport Pathways through Human P-Glycoprotein.

PubMed

McCormick, James W; Vogel, Pia D; Wise, John G

2015-07-21

P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

Particle detector spatial resolution

DOEpatents

Perez-Mendez, V.

1992-12-15

Method and apparatus for producing separated columns of scintillation layer material, for use in detection of X-rays and high energy charged particles with improved spatial resolution is disclosed. A pattern of ridges or projections is formed on one surface of a substrate layer or in a thin polyimide layer, and the scintillation layer is grown at controlled temperature and growth rate on the ridge-containing material. The scintillation material preferentially forms cylinders or columns, separated by gaps conforming to the pattern of ridges, and these columns direct most of the light produced in the scintillation layer along individual columns for subsequent detection in a photodiode layer. The gaps may be filled with a light-absorbing material to further enhance the spatial resolution of the particle detector. 12 figs.

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

Particle detector spatial resolution

DOEpatents

Perez-Mendez, Victor

1992-01-01

Method and apparatus for producing separated columns of scintillation layer material, for use in detection of X-rays and high energy charged particles with improved spatial resolution. A pattern of ridges or projections is formed on one surface of a substrate layer or in a thin polyimide layer, and the scintillation layer is grown at controlled temperature and growth rate on the ridge-containing material. The scintillation material preferentially forms cylinders or columns, separated by gaps conforming to the pattern of ridges, and these columns direct most of the light produced in the scintillation layer along individual columns for subsequent detection in a photodiode layer. The gaps may be filled with a light-absorbing material to further enhance the spatial resolution of the particle detector.

Stressor-layer-induced elastic strain sharing in SrTiO 3 complex oxide sheets

DOE PAGES

Tilka, J. A.; Park, J.; Ahn, Y.; ...

2018-02-26

A precisely selected elastic strain can be introduced in submicron-thick single-crystal SrTiO 3 sheets using a silicon nitride stressor layer. A conformal stressor layer deposited using plasma-enhanced chemical vapor deposition produces an elastic strain in the sheet consistent with the magnitude of the nitride residual stress. Synchrotron x-ray nanodiffraction reveals that the strain introduced in the SrTiO 3 sheets is on the order of 10 -4, matching the predictions of an elastic model. Using this approach to elastic strain sharing in complex oxides allows the strain to be selected within a wide and continuous range of values, an effect notmore » achievable in heteroepitaxy on rigid substrates.« less

Stressor-layer-induced elastic strain sharing in SrTiO 3 complex oxide sheets

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

Tilka, J. A.; Park, J.; Ahn, Y.

A precisely selected elastic strain can be introduced in submicron-thick single-crystal SrTiO 3 sheets using a silicon nitride stressor layer. A conformal stressor layer deposited using plasma-enhanced chemical vapor deposition produces an elastic strain in the sheet consistent with the magnitude of the nitride residual stress. Synchrotron x-ray nanodiffraction reveals that the strain introduced in the SrTiO 3 sheets is on the order of 10 -4, matching the predictions of an elastic model. Using this approach to elastic strain sharing in complex oxides allows the strain to be selected within a wide and continuous range of values, an effect notmore » achievable in heteroepitaxy on rigid substrates.« less

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.

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

PubMed

Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

2009-09-01

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

Insights into molecular mechanisms of drug metabolism dysfunction of human CYP2C9*30

PubMed Central

Louet, Maxime; Labbé, Céline M.; Aono, Cassiano M.; Homem-de-Mello, Paula; Villoutreix, Bruno O.

2018-01-01

Cytochrome P450 2C9 (CYP2C9) metabolizes about 15% of clinically administrated drugs. The allelic variant CYP2C9*30 (A477T) is associated to diminished response to the antihypertensive effects of the prodrug losartan and affected metabolism of other drugs. Here, we investigated molecular mechanisms involved in the functional consequences of this amino-acid substitution. Molecular dynamics (MD) simulations performed for the active species of the enzyme (heme in the Compound I state), in the apo or substrate-bound state, and binding energy analyses gave insights into altered protein structure and dynamics involved in the defective drug metabolism of human CYP2C9.30. Our data revealed an increased rigidity of the key Substrate Recognition Sites SRS1 and SRS5 and shifting of the β turn 4 of SRS6 toward the helix F in CYP2C9.30. Channel and binding substrate dynamics analyses showed altered substrate channel access and active site accommodation. These conformational and dynamic changes are believed to be involved in the governing mechanism of the reduced catalytic activity. An ensemble of representative conformations of the WT and A477T mutant properly accommodating drug substrates were identified, those structures can be used for prediction of new CYP2C9 and CYP2C9.30 substrates and drug-drug interactions. PMID:29746595

Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

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

Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.

2010-12-08

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pocketsmore » that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.« less

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

PubMed Central

Nallamsetty, Sreedevi; Waugh, David S.

2007-01-01

Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its “open” and “closed” conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement. PMID:17964542

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs

PubMed Central

Blachly, Patrick G.; de Oliveira, César A. F.; Williams, Sarah L.; McCammon, J. Andrew

2013-01-01

The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe4S4]2+ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts. PMID:24367248

Photoinduced electron transfer through peptide-based self-assembled monolayers chemisorbed on gold electrodes: directing the flow-in and flow-out of electrons through peptide helices.

PubMed

Venanzi, Mariano; Gatto, Emanuela; Caruso, Mario; Porchetta, Alessandro; Formaggio, Fernando; Toniolo, Claudio

2014-08-21

Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.

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.

Molecular basis of activation of endopeptidase activity of botulinum neurotoxin type E.

PubMed

Kukreja, Roshan V; Sharma, Shashi K; Singh, Bal Ram

2010-03-23

Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine, thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active dichain form of the toxin. Nicking of the single chain BoNT/E to the dichain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 degrees C the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction-induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.

Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

PubMed

Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

2013-08-21

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

PubMed

Schmitt, Kyle C; Mamidyala, Sreeman; Biswas, Swati; Dutta, Aloke K; Reith, Maarten E A

2010-03-01

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies*

PubMed Central

Liu, Yao; Rodrigues, João P. G. L. M.; Bonvin, Alexandre M. J. J.; Zaal, Esther A.; Berkers, Celia R.; Heger, Michal; Gawarecka, Katarzyna; Swiezewska, Ewa; Breukink, Eefjan; Egmond, Maarten R.

2016-01-01

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide. PMID:27226570

Electromagnetic characterization of conformal antennas

NASA Technical Reports Server (NTRS)

Volakis, John L.; Kempel, Leo C.; Alexanian, Angelos; Jin, J. M.; Yu, C. L.; Woo, Alex C.

1992-01-01

The ultimate objective of this project is to develop a new technique which permits an accurate simulation of microstrip patch antennas or arrays with various feed, superstrate and/or substrate configurations residing in a recessed cavity whose aperture is planar, cylindrical or otherwise conformed to the substructure. The technique combines the finite element and boundary integral methods to formulate a system suitable for solution via the conjugate gradient method in conjunction with the fast Fourier transform. The final code is intended to compute both scattering and radiation patterns of the structure with an affordable memory demand. With upgraded capabilities, the four included papers examined the radar cross section (RCS), input impedance, gain, and resonant frequency of several rectangular configurations using different loading and substrate/superstrate configurations.

Carbon nanotube active-matrix backplanes for conformal electronics and sensors.

PubMed

Takahashi, Toshitake; Takei, Kuniharu; Gillies, Andrew G; Fearing, Ronald S; Javey, Ali

2011-12-14

In this paper, we report a promising approach for fabricating large-scale flexible and stretchable electronics using a semiconductor-enriched carbon nanotube solution. Uniform semiconducting nanotube networks with superb electrical properties (mobility of ∼20 cm2 V(-1) s(-1) and ION/IOFF of ∼10(4)) are obtained on polyimide substrates. The substrate is made stretchable by laser cutting a honeycomb mesh structure, which combined with nanotube-network transistors enables highly robust conformal electronic devices with minimal device-to-device stochastic variations. The utility of this device concept is demonstrated by fabricating an active-matrix backplane (12×8 pixels, physical size of 6×4 cm2) for pressure mapping using a pressure sensitive rubber as the sensor element.

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.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

PubMed Central

Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

2010-01-01

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

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.

Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase†

PubMed Central

Deng, Hua; Vu, Dung V.; Clinch, Keith; Desamero, Ruel; Dyer, R. Brian; Callender, Robert

2011-01-01

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme. PMID:21568287

Tailored nanoporous coatings fabricated on conformable polymer substrates.

PubMed

Poxson, David J; Mont, Frank W; Cho, Jaehee; Schubert, E Fred; Siegel, Richard W

2012-11-01

Nanoporous coatings have become the subject of intense investigation, in part because they have been shown to have unique and tailorable physical properties that can depart greatly from their dense or macroscopic counterparts. Nanoporous coatings are frequently fabricated utilizing oblique-angle or glancing-angle physical vapor-phase deposition techniques. However, a significant limitation for such coatings exists; they are almost always deposited on smooth and rigid planar substrates, such as silicon and glass. This limitation greatly constrains the applicability, tailorability, functionality and even the economic viability, of such nanoporous coatings. Here, we report our findings on nanoporous/polymer composite systems (NPCS) fabricated by utilizing oblique-angle electron-beam methodology. These unique composite systems exhibit several favorable characteristics, namely, (i) fine-tuned control over coating nanoporosity and thickness, (ii) excellent adhesion between the nanoporous coating and polymer substrate, (iii) the ability to withstand significant and repeated bending, and (iv) the ability to be molded conformably on two and three-dimensional surfaces while closely retaining the composite system's designed nanoporous film structure and, hence, properties.

Visualizing chaperone-assisted protein folding

DOE PAGES

Horowitz, Scott; Salmon, Loïc; Koldewey, Philipp; ...

2016-05-30

We present that challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperonemore » Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.« less

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

Promotion of pro-osteogenic responses by a bioactive ceramic coating.

PubMed

Aniket; Young, Amy; Marriott, Ian; El-Ghannam, Ahmed

2012-12-01

The objective of this study was to analyze the responses of bone-forming osteoblasts to Ti-6Al-4V implant material coated with silica-calcium phosphate nanocomposite (SCPC50). Osteoblast differentiation at the interface with SCPC50-coated Ti-6Al-4V was correlated to the adsorption of high amount of serum proteins, high surface affinity to fibronectin, Ca uptake from and P and Si release into the medium. SCPC50-coated Ti-6Al-4V adsorbed significantly more serum protein (p < 0.05) than control uncoated substrates. Moreover, Western blot analysis showed that the SCPC50 coating had a high affinity for serum fibronectin. Protein conformation analyses by FTIR showed that the ratio of the area under the peak for amide I/amide II bands was significantly higher (p < 0.05) on the surface of SCPC50-coated substrates than that on the surface of the control uncoated substrates. Moreover, ICP - OES analyses indicated that SCPC50-coated substrates withdrew Ca ions from, and released P and Si ions into, the tissue culture medium, respectively. In conjunction with the favorable protein adsorption and modifications in medium composition, MC3T3-E1 osteoblast-like cells attached to SCPC50-coated substrates expressed 10-fold higher level of mRNA encoding osteocalcin and had significantly higher production of osteopontin and osteocalcin proteins than cells attached to the uncoated Ti-6A1-4V substrates. In addition, osteoblast-like cells attached to the SCPC50-coated substrates produced significantly lower levels of the inflammatory and osteoclastogenic cytokines, IL-6, IL-12p40, and RANKL than those attached to uncoated Ti-6Al-4V substrates. These results suggest that SCPC50 coating could enhance bone integration with orthopedic and maxillofacial implants while minimizing the induction of inflammatory bone cell responses. Copyright © 2012 Wiley Periodicals, Inc.

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.

Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

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

González-Páez, Gonzalo E.; Wolan, Dennis W.

2012-09-05

Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50}more » values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.« less

Microfluidics on compliant substrates: recent developments in foldable and bendable devices and system packaging

NASA Astrophysics Data System (ADS)

Gray, Bonnie L.

2012-04-01

Microfluidics is revolutionizing laboratory methods and biomedical devices, offering new capabilities and instrumentation in multiple areas such as DNA analysis, proteomics, enzymatic analysis, single cell analysis, immunology, point-of-care medicine, personalized medicine, drug delivery, and environmental toxin and pathogen detection. For many applications (e.g., wearable and implantable health monitors, drug delivery devices, and prosthetics) mechanically flexible polymer devices and systems that can conform to the body offer benefits that cannot be achieved using systems based on conventional rigid substrate materials. However, difficulties in implementing active devices and reliable packaging technologies have limited the success of flexible microfluidics. Employing highly compliant materials such as PDMS that are typically employed for prototyping, we review mechanically flexible polymer microfluidic technologies based on free-standing polymer substrates and novel electronic and microfluidic interconnection schemes. Central to these new technologies are hybrid microfabrication methods employing novel nanocomposite polymer materials and devices. We review microfabrication methods using these materials, along with demonstrations of example devices and packaging schemes that employ them. We review these recent developments and place them in the context of the fields of flexible microfluidics and conformable systems, and discuss cross-over applications to conventional rigid-substrate microfluidics.

Soft lubrication: The elastohydrodynamics of nonconforming and conforming contacts

NASA Astrophysics Data System (ADS)

Skotheim, J. M.; Mahadevan, L.

2005-09-01

We study the lubrication of fluid-immersed soft interfaces and show that elastic deformation couples tangential and normal forces and thus generates lift. We consider materials that deform easily, due to either geometry (e.g., a shell) or constitutive properties (e.g., a gel or a rubber), so that the effects of pressure and temperature on the fluid properties may be neglected. Four different system geometries are considered: a rigid cylinder moving parallel to a soft layer coating a rigid substrate; a soft cylinder moving parallel to a rigid substrate; a cylindrical shell moving parallel to a rigid substrate; and finally a cylindrical conforming journal bearing coated with a thin soft layer. In addition, for the particular case of a soft layer coating a rigid substrate, we consider both elastic and poroelastic material responses. For all these cases, we find the same generic behavior: there is an optimal combination of geometric and material parameters that maximizes the dimensionless normal force as a function of the softness parameter η =hydrodynamicpressure/elasticstiffness=surfacedeflection/gapthickness, which characterizes the fluid-induced deformation of the interface. The corresponding cases for a spherical slider are treated using scaling concepts.

Method for producing highly conformal transparent conducting oxides

DOEpatents

Elam, Jeffrey W.; Mane, Anil U.

2016-07-26

A method for forming a transparent conducting oxide product layer. The method includes use of precursors, such as tetrakis-(dimethylamino) tin and trimethyl indium, and selected use of dopants, such as SnO and ZnO for obtaining desired optical, electrical and structural properties for a highly conformal layer coating on a substrate. Ozone was also input as a reactive gas which enabled rapid production of the desired product layer.

Insights on Na(+) binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM.

PubMed

Song, Jianing; Ji, Changge; Zhang, John Z H

2014-02-01

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+-substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild-type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na(+) ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di-Na(+) binding state is likely more efficient for conformational changes of NorM_VC toward the inward-facing conformation than single-Na(+) binding state. The observation of two Na(+) binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na(+) binding and enrich our knowledge of the transport mechanism of secondary transporters. Copyright © 2013 Wiley Periodicals, Inc.

Enhanced Pb Absorption by Hordeum vulgare L. and Helianthus annuus L. Plants Inoculated with an Arbuscular Mycorrhizal Fungi Consortium.

PubMed

Arias, Milton Senen Barcos; Peña-Cabriales, Juan José; Alarcón, Alejandro; Maldonado Vega, María

2015-01-01

The effect of an arbuscular mycorrhizal fungi (AMF) consortium conformed by (Glomus intraradices, Glomus albidum, Glomus diaphanum, and Glomus claroideum) on plant growth and absorption of Pb, Fe, Na, Ca, and (32)P in barley (Hordeum vulgare L.) and sunflower (Helianthus annuus L.) plants was evaluated. AMF-plants and controls were grown in a substrate amended with powdered Pb slag at proportions of 0, 10, 20, and 30% v/v equivalent to total Pb contents of 117; 5,337; 13,659, and 19,913 mg Pb kg(-1) substrate, respectively. Mycorrhizal root colonization values were 70, 94, 98, and 90%, for barley and 91, 97, 95, and 97%, for sunflower. AMF inoculum had positive repercussions on plant development of both crops. Mycorrhizal barley absorbed more Pb (40.4 mg Pb kg(-1)) shoot dry weight than non-colonized controls (26.5 mg Pb kg(-1)) when treated with a high Pb slag dosage. This increase was higher in roots than shoots (650.0 and 511.5 mg Pb kg(-1) root dry weight, respectively). A similar pattern was found in sunflower. Plants with AMF absorbed equal or lower amounts of Fe, Na and Ca than controls. H. vulgare absorbed more total P (1.0%) than H. annuus (0.9%). The arbuscular mycorrizal consortium enhanced Pb extraction by plants.

Investigations in sonication-induced intensification of crude glycerol fermentation to dihydroxyacetone by free and immobilized Gluconobacter oxydans.

PubMed

Dikshit, Pritam Kumar; Kharmawlong, Gracel Joe; Moholkar, Vijayanand S

2018-05-01

This study reports crude glycerol fermentation by G. oxydans for dihydroxyacetone (DHA) production, and intensification of fermentation with sonication. Fermentation was carried out using both free and immobilized cells (on polyurethane foam support) for initial glycerol concentrations of 20, 30 and 50 g/L. Sonication at 20% duty cycle enhanced glycerol consumption by 60-84% with no significant change in cell morphology. Lesser DHA yield in crude glycerol fermentation was attributed to possible formation of inhibitory products. Slight reduction in DHA yield for initial glycerol concentration of 50 g/L was attributed to substrate inhibition. Higher DHA productivity was obtained for immobilized cells. Circular dichroism analysis of intracellular proteins obtained from ultrasound-treated G. oxydans revealed significant reduction in α-helix and β-sheet content. These conformational changes in protein structure could augment activity of intracellular glycerol dehydrogenase, which is manifested in terms of enhanced metabolism of glycerol by G. oxydans. Copyright © 2018 Elsevier Ltd. All rights reserved.

An ordered array of hierarchical spheres for surface-enhanced Raman scattering detection of traces of pesticide

NASA Astrophysics Data System (ADS)

Hu, Xiaoye; Zheng, Peng; Meng, Guowen; Huang, Qing; Zhu, Chuhong; Han, Fangming; Huang, Zhulin; Li, Zhongbo; Wang, Zhaoming; Wu, Nianqiang

2016-09-01

An ordered array of hierarchically-structured core-nanosphere@space-layer@shell-nanoparticles has been fabricated for surface-enhanced Raman scattering (SERS) detection. To fabricate this hierarchically-structured chip, a long-range ordered array of Au/Ag-nanospheres is first patterned in the nano-bowls on the planar surface of ordered nanoporous anodic titanium oxide template. A ultra-thin alumina middle space-layer is then conformally coated on the Au/Ag-nanospheres, and Ag-nanoparticles are finally deposited on the surface of the alumina space-layer to form an ordered array of Au/Ag-nanosphere@Al2O3-layer@Ag-nanoparticles. Finite-difference time-domain simulation shows that SERS hot spots are created between the neighboring Ag-nanoparticles. The ordered array of hierarchical nanostructures is used as the SERS-substrate for a trial detection of methyl parathion (a pesticide) in water and a limit of detection of 1 nM is reached, indicating its promising potential in rapid monitoring of organic pollutants in aquatic environment.

The dynamics of camphor in the cytochrome P450 CYP101D2

PubMed Central

Vohra, Shabana; Musgaard, Maria; Bell, Stephen G; Wong, Luet-Lok; Zhou, Weihong; Biggin, Philip C

2013-01-01

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate-free) and camphor-soaked forms have open conformations. Furthermore, two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure, one being located in the access channel and the other in a cavity on the surface near the F-helix side of the F-G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor-bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor-bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit. PMID:23832606

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.

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

The Cytoplasmic Permeation Pathway of Neurotransmitter Transporters†

PubMed Central

Rudnick, Gary

2011-01-01

Ion-coupled solute transporters are responsible for transporting nutrients, ions and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes required for solute transport in this family. The first structure in this family showed the bacterial amino acid transporter LeuT, which is homologous to neurotransmitter transporters, in an extracellularly-oriented conformation with a molecule of leucine occluded at the substrate site. Studies with the mammalian serotonin transporter identified positions, buried in the LeuT structure, that defined a potential pathway leading from the cytoplasm to the substrate binding site. Modeling studies utilized an inverted structural repeat within the LeuT crystal structure to predict the conformation of LeuT in which the cytoplasmic permeation pathway, consisting of positions identified in SERT, was open for substrate diffusion to the cytoplasm. From the difference between the model and the crystal structures, a simple “rocking bundle” mechanism was proposed, in which a 4-helix bundle changed its orientation with respect to the rest of the protein to close the extracellular pathway and open the cytoplasmic one. Subsequent crystal structures from structurally related proteins provide evidence supporting this model for transport. PMID:21774491

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

PubMed

Edwards, J Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle Nee; French, Alfred D; Condon, Brian D

2018-03-13

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity ( K m ) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased K m observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency ( k cat / K m ), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Characteristics of a Linearly Tapered Slot Antenna (LTSA) Conformed Longitudinally Around a Cylinder

NASA Technical Reports Server (NTRS)

Jordan, Jennifer L.; Ponchak, George E.; Tavassolian, Negar; Tentzeris, Manos M.

2007-01-01

The family of tapered slot antennas (TSA s) is suitable for numerous applications. Their ease of fabrication, wide bandwidth, and high gain make them desirable for military and commercial systems. Fabrication on thin, flexible substrates allows the TSA to be conformed over a given body, such as an aircraft wing or a piece of clothing for wearable networks. Previously, a Double Exponentially Tapered Slot Antenna (DETSA) was conformed around an exponential curvature, which showed that the main beam skewed towards the direction of curvature. This paper presents a Linearly Tapered Slot Antenna (LTSA) conformed longitudinally around a cylinder. Measured and simulated radiation patterns and the direction of maximum H co-polarization (Hco) as a function of the cylinder radius are presented.

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.

Using THz Spectroscopy, Evolutionary Network Analysis Methods, and MD Simulation to Map the Evolution of Allosteric Communication Pathways in c-Type Lysozymes.

PubMed

Woods, Kristina N; Pfeffer, Juergen

2016-01-01

It is now widely accepted that protein function is intimately tied with the navigation of energy landscapes. In this framework, a protein sequence is not described by a distinct structure but rather by an ensemble of conformations. And it is through this ensemble that evolution is able to modify a protein's function by altering its landscape. Hence, the evolution of protein functions involves selective pressures that adjust the sampling of the conformational states. In this work, we focus on elucidating the evolutionary pathway that shaped the function of individual proteins that make-up the mammalian c-type lysozyme subfamily. Using both experimental and computational methods, we map out specific intermolecular interactions that direct the sampling of conformational states and accordingly, also underlie shifts in the landscape that are directly connected with the formation of novel protein functions. By contrasting three representative proteins in the family we identify molecular mechanisms that are associated with the selectivity of enhanced antimicrobial properties and consequently, divergent protein function. Namely, we link the extent of localized fluctuations involving the loop separating helices A and B with shifts in the equilibrium of the ensemble of conformational states that mediate interdomain coupling and concurrently moderate substrate binding affinity. This work reveals unique insights into the molecular level mechanisms that promote the progression of interactions that connect the immune response to infection with the nutritional properties of lactation, while also providing a deeper understanding about how evolving energy landscapes may define present-day protein function. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

PubMed Central

Gordon, Kerry; Nesterovitch, Andrew B.; Lünsdorf, Heinrich; Chen, Zhenlong; Castellon, Maricela; Popova, Isolda A.; Kalinin, Sergey; Mendonca, Emma; Petukhov, Pavel A.; Schwartz, David E.

2011-01-01

Background Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. Methodology/Principal Findings HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase). PMID:21998728

Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2

PubMed Central

2017-01-01

The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate. PMID:28884161

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

PubMed Central

Amyot, Whitney M.; deJesus, Dennise

2013-01-01

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536

Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1).

PubMed

Asciutto, Eliana K; Pochapsky, Thomas C

2018-04-27

Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H- 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution.

PubMed

Hu, Chen; Fang, Jianwen; Borchardt, Ronald T; Schowen, Richard L; Kuczera, Krzysztof

2008-04-01

S-Adenosyl-L-homocysteine hydrolase (SAHH) is an enzyme regulating intracellular methylation reactions. The homotetrameric SAHH exists in an open conformation in absence of substrate, while enzyme:inhibitor complexes crystallize in the closed conformation, in which the ligands are engulfed by the protein due to an 18 degrees domain reorientation within each of the four subunits. We present a microscopic description of the structure and dynamics of the substrate-free, NAD(+)-bound SAHH in solution, based on a 15-ns molecular dynamics simulation in explicit solvent. In the trajectory, the four cofactor-binding domains formed a relatively rigid core with structure very similar to the crystal conformation. The four substrate-binding domains, located at the protein exterior, also retained internal structures similar to the crystal, while undergoing large amplitude rigid-body reorientations. The trajectory domain motions exhibited two interesting properties. First, within each subunit the domains fluctuated between open and closed conformations, while at the tetramer level 80% of the domain motions were perpendicular to the direction of the open-to-closed structural transition. Second, the domain reorientations in solution could be represented as a sum of two components, faster, with 20-50 ps correlation time and 3-4 degrees amplitude, and slower, with 8-23 ns correlation time and amplitude of 14-22 degrees . The faster motion is similar to the 1.5 cm(-1) frequency hinge-bending vibrations found in our recent normal mode analysis (Wang et al., Biochemistry 2005;44:7228-7239). The slower motion agrees with fluorescence anisotropy decay measurements, which detected a 10-20 ns domain reorientation of ca. 26 degrees amplitude in the substrate-free enzyme (Wang et al., Biochemistry 2006;45:7778-7786). Our simulations are thus in excellent agreement with experimental data. The simulations allow us to assign the observed nanosecond fluorescence anisotropy signal to fluctuations in domain orientations, and indicate that the microscopic mechanism of the motion involves rotational diffusion within a cone of 10-20 degrees . Overall, our simulation results complement the existing experimental data and provide important new insights into SAHH domain motions in solution, which play a crucial role in the catalytic mechanism of SAHH. (c) 2007 Wiley-Liss, Inc.

Understanding the sorption and biotransformation of organic micropollutants in innovative biological wastewater treatment technologies.

PubMed

Alvarino, T; Suarez, S; Lema, J; Omil, F

2018-02-15

New technologies for wastewater treatment have been developed in the last years based on the combination of biological reactors operating under different redox conditions. Their efficiency in the removal of organic micropollutants (OMPs) has not been clearly assessed yet. This review paper is focussed on understanding the sorption and biotransformation of a selected group of 17 OMPs, including pharmaceuticals, hormones and personal care products, during biological wastewater treatment processes. Apart from considering the role of "classical" operational parameters, new factors such as biomass conformation and particle size, upward velocity applied or the addition of adsorbents have been considered. It has been found that the OMP removal by sorption not only depends on their physico-chemical characteristics and other parameters, such as the biomass conformation and particle size, or some operational conditions also relevant. Membrane biological reactors (MBR), have shown to enhance sorption and biotransformation of some OMPs. The same applies to technologies bases on direct addition of activated carbon in bioreactors. The OMP biotransformation degree and pathway is mainly driven by the redox potential and the primary substrate activity. The combination of different redox potentials in hybrid reactor systems can significantly enhance the overall OMP removal efficiency. Sorption and biotransformation can be synergistically promoted in biological reactors by the addition of activated carbon. The deeper knowledge of the main parameters influencing OMP removal provided by this review will allow optimizing the biological processes in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

Flexible digital x-ray technology for far-forward remote diagnostic and conformal x-ray imaging applications

NASA Astrophysics Data System (ADS)

Smith, Joseph; Marrs, Michael; Strnad, Mark; Apte, Raj B.; Bert, Julie; Allee, David; Colaneri, Nicholas; Forsythe, Eric; Morton, David

2013-05-01

Today's flat panel digital x-ray image sensors, which have been in production since the mid-1990s, are produced exclusively on glass substrates. While acceptable for use in a hospital or doctor's office, conventional glass substrate digital x-ray sensors are too fragile for use outside these controlled environments without extensive reinforcement. Reinforcement, however, significantly increases weight, bulk, and cost, making them impractical for far-forward remote diagnostic applications, which demand rugged and lightweight x-ray detectors. Additionally, glass substrate x-ray detectors are inherently rigid. This limits their use in curved or bendable, conformal x-ray imaging applications such as the non-destructive testing (NDT) of oil pipelines. However, by extending low-temperature thin-film transistor (TFT) technology previously demonstrated on plastic substrate- based electrophoretic and organic light emitting diode (OLED) flexible displays, it is now possible to manufacture durable, lightweight, as well as flexible digital x-ray detectors. In this paper, we discuss the principal technical approaches used to apply flexible display technology to two new large-area flexible digital x-ray sensors for defense, security, and industrial applications and demonstrate their imaging capabilities. Our results include a 4.8″ diagonal, 353 x 463 resolution, flexible digital x-ray detector, fabricated on a 6″ polyethylene naphthalate (PEN) plastic substrate; and a larger, 7.9″ diagonal, 720 x 640 resolution, flexible digital x-ray detector also fabricated on PEN and manufactured on a gen 2 (370 x 470 mm) substrate.

Adsorption of human serum albumin: Dependence on molecular architecture of the oppositely charged surface

NASA Astrophysics Data System (ADS)

Sukhishvili, Svetlana A.; Granick, Steve

1999-05-01

We contrast the adsorption of human serum albumin (HSA) onto two solid substrates previously primed with the same polyelectrolyte of net opposite charge to form one of two alternative structures: randomly adsorbed polymer and the "brush" configuration. These structures were formed either by the adsorption of quaternized poly-4-vinylpyridine (QPVP) or by end-grafting QPVP chains of the same chemical makeup and the same molecular weight to surfaces onto which QPVP segments did not adsorb. The adsorption of HSA was quantified by using Fourier transform infrared spectroscopy in attenuated total reflection (FTIR-ATR). The two substrates showed striking differences with regard to HSA adsorption. First, the brush substrate induced lesser perturbations in the secondary structure of the adsorbed HSA, reflecting easier conformational adjustment for longer free segments of polyelectrolyte upon binding with the protein. Second, the penetration of HSA into the brush substrate was kinetically retarded relative to the randomly adsorbed polymer, probably due to both pore size restriction and electrostatic sticking between charged groups of HSA and QPVP molecules. Third, release of HSA from the adsorbed layer, as the ionic strength was increased from a low level up to the high level of 1 M NaCl, was largely inhibited for the brush substrate, but occurred easily and rapidly for the substrate with statistically adsorbed QPVP chains. Finally, even after addition of a strong polymeric adsorption competitor (sodium polystyrene sulfonate), HSA remained trapped within a brush substrate though it desorbed slowly from the preadsorbed QPVP layer. This method to produce irreversible trapping of the protein within a brush substrate without major conformational change may find application in biosensor design.

Method for preparing ultraflat, atomically perfect areas on large regions of a crystal surface by heteroepitaxy deposition

DOEpatents

El Gabaly, Farid; Schmid, Andreas K.

2013-03-19

A novel method of forming large atomically flat areas is described in which a crystalline substrate having a stepped surface is exposed to a vapor of another material to deposit a material onto the substrate, which material under appropriate conditions self arranges to form 3D islands across the substrate surface. These islands are atomically flat at their top surface, and conform to the stepped surface of the substrate below at the island-substrate interface. Thereafter, the deposited materials are etched away, in the etch process the atomically flat surface areas of the islands transferred to the underlying substrate. Thereafter the substrate may be cleaned and annealed to remove any remaining unwanted contaminants, and eliminate any residual defects that may have remained in the substrate surface as a result of pre-existing imperfections of the substrate.

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

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.

Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights.

PubMed

Chetty, Sarentha; Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

2016-01-01

The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor-protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔGbind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by retaining its key pharmacophore features will be the way forward in the search for novel protease inhibitors against multi-drug resistant strains.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor.

PubMed

Zhang, Xirui; Daaboul, George G; Spuhler, Philipp S; Dröge, Peter; Ünlü, M Selim

2016-03-14

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

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

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study.

PubMed

Huang, Rui; Ripstein, Zev A; Augustyniak, Rafal; Lazniewski, Michal; Ginalski, Krzysztof; Kay, Lewis E; Rubinstein, John L

2016-07-19

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.

A substrate selectivity and inhibitor design lesson from the PDE10-cAMP crystal structure: a computational study.

PubMed

Lau, Justin Kai-Chi; Li, Xiao-Bo; Cheng, Yuen-Kit

2010-04-22

Phosphodiesterases (PDEs) catalyze the hydrolysis of second messengers cAMP and cGMP in regulating many important cellular signals and have been recognized as important drug targets. Experimentally, a range of specificity/selectivity toward cAMP and cGMP is well-known for the individual PDE families. The study reported here reveals that PDEs might also exhibit selectivity toward conformations of the endogenous substrates cAMP and cGMP. Molecular dynamics simulations and free energy study have been applied to study the binding of the cAMP torsional conformers about the glycosyl bond in PDE10A2. The computational results elucidated that PDE10A2 is energetically more favorable in complex with the syn cAMP conformer (as reported in the crystal structure) and the binding of anti cAMP to PDE10A2 would lead to either a nonreactive configuration or significant perturbation on the catalytic pocket of the enzyme. This experimentally inaccessible information provides important molecular insights for the development of effective PDE10 ligands.

Helical unwinding and side-chain unlocking unravel the outward open conformation of the melibiose transporter

NASA Astrophysics Data System (ADS)

Wang, Li-Ying; Ravi, Vidhya M.; Leblanc, Gérard; Padrós, Esteve; Cladera, Josep; Perálvarez-Marín, Alex

2016-09-01

Molecular dynamics simulations have been used to study the alternate access mechanism of the melibiose transporter from Escherichia coli. Starting from the outward-facing partially occluded form, 2 out of 12 simulations produced an outward full open form and one partially open, whereas the rest yielded fully or partially occluded forms. The shape of the outward-open form resembles other outward-open conformations of secondary transporters. During the transporter opening, conformational changes in some loops are followed by changes in the periplasm region of transmembrane helix 7. Helical curvature relaxation and unlocking of hydrophobic and ionic locks promote the outward opening of the transporter making accessible the substrate binding site. In particular, FRET studies on mutants of conserved aromatic residues of extracellular loop 4 showed lack of substrate binding, emphasizing the importance of this loop for making crucial interactions that control the opening of the periplasmic side. This study indicates that the alternate access mechanism for the melibiose transporter fits better into a flexible gating mechanism rather than the archetypical helical rigid-body rocker-switch mechanism.

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

Mechanisms of chain adsorption on porous substrates and critical conditions of polymer chromatography.

PubMed

Cimino, Richard T; Rasmussen, Christopher J; Brun, Yefim; Neimark, Alexander V

2016-11-01

Polymer adsorption is a ubiquitous phenomenon with numerous technological and healthcare applications. The mechanisms of polymer adsorption on surfaces and in pores are complex owing to a competition between various entropic and enthalpic factors. Due to adsorption of monomers to the surface, the chain gains in enthalpy yet loses in entropy because of confining effects. This competition leads to the existence of critical conditions of adsorption when enthalpy gain and entropy loss are in balance. The critical conditions are controlled by the confining geometry and effective adsorption energy, which depends on the solvent composition and temperature. This phenomenon has important implications in polymer chromatography, since the retention at the critical point of adsorption (CPA) is chain length independent. However, the mechanisms of polymer adsorption in pores are poorly understood and there is an ongoing discussion in the theoretical literature about the very existence of CPA for polymer adsorption on porous substrates. In this work, we examine the mechanisms of chain adsorption on a model porous substrate using Monte Carlo (MC) simulations. We distinguish three adsorption mechanisms depending on the chain location: on external surface, completely confined in pores, and also partially confined in pores in so-called "flower" conformations. The free energies of different conformations of adsorbed chains are calculated by the incremental gauge cell MC method that allows one to determine the partition coefficient as a function of the adsorption potential, pore size, and chain length. We confirm the existence of the CPA for chain length independent separation on porous substrates, which is explained by the dominant contributions of the chain adsorption at the external surface, in particular in flower conformations. Moreover, we show that the critical conditions for porous and nonporous substrates are identical and depend only on the surface chemistry. The theoretical results are confirmed by comparison with experimental data on chromatographic separation of a series of linear polystyrenes. Copyright © 2016 Elsevier Inc. All rights reserved.

AFM study of adsorption of protein A on a poly(dimethylsiloxane) surface

NASA Astrophysics Data System (ADS)

Yu, Ling; Lu, Zhisong; Gan, Ye; Liu, Yingshuai; Li, Chang Ming

2009-07-01

In this paper, the morphology and kinetics of adsorption of protein A on a PDMS surface is studied by AFM. The results of effects of pH, protein concentration and contact time of the adsorption reveal that the morphology of adsorbed protein A is significantly affected by pH and adsorbed surface concentration, in which the pH away from the isoelectric point (IEP) of protein A could produce electrical repulsion to change the protein conformation, while the high adsorbed surface protein volume results in molecular networks. Protein A can form an adsorbed protein film on PDMS with a maximum volume of 2.45 × 10-3 µm3. This work enhances our fundamental understanding of protein A adsorption on PDMS, a frequently used substrate component in miniaturized immunoassay devices.

Conformational dynamics of abasic DNA upon interactions with AP endonuclease 1 revealed by stopped-flow fluorescence analysis.

PubMed

Kanazhevskaya, Lyubov Yu; Koval, Vladimir V; Vorobjev, Yury N; Fedorova, Olga S

2012-02-14

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.

SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

PubMed

Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

2009-07-01

Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides.

PubMed Central

Komatsu, Y; Kumagai, I; Ohtsuka, E

1999-01-01

We introduced 4-thio- ((4S)U), 2-thio- ((2S)U), 4- O -methyluridine ((4Me)U) and cytidine substitutions for U+2, which is an important base for cleavage in a substrate RNA. Oligonucleotides containing 4-thio- and 4- O -methyluridine were prepared by a new convenient post-synthetic modification method using a 4- O - p -nitrophenyl-uridine derivative. A hairpin ribozyme cleaved the substrate RNA with either C+2, (4S)U+2 or (4Me)U+2 at approximately 14-, 6- and 4-fold lower rates, respectively, than that of the natural substrate. In contrast, the substrate with a (2S)U+2 was cleaved with the same activity as the natural substrate. These results suggest that the O4 of U+2 is involved in hydrogen bonding at loop A, but the O2 of U+2 is not recognized by the active residues. Circular dichroism data of the ribozymes containing (4S)U+2 and (2S)U+2, as well as the susceptibility of the thiocarbonyl group to hydrogen peroxide, suggest that a conformational change of U+2 occurs during the domain docking in the cleavage reaction. We propose here the conformational change of U+2 from the ground state to the active molecule during the reaction. PMID:10536137

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group.

PubMed

Sulzenbacher, G; Driguez, H; Henrissat, B; Schülein, M; Davies, G J

1996-12-03

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

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

System of fabricating a flexible electrode array

DOEpatents

Krulevitch, Peter; Polla, Dennis L.; Maghribi, Mariam N.; Hamilton, Julie; Humayun, Mark S.; Weiland, James D.

2010-10-12

An image is captured or otherwise converted into a signal in an artificial vision system. The signal is transmitted to the retina utilizing an implant. The implant consists of a polymer substrate made of a compliant material such as poly(dimethylsiloxane) or PDMS. The polymer substrate is conformable to the shape of the retina. Electrodes and conductive leads are embedded in the polymer substrate. The conductive leads and the electrodes transmit the signal representing the image to the cells in the retina. The signal representing the image stimulates cells in the retina.

System of fabricating a flexible electrode array

DOEpatents

Krulevitch, Peter [Pleasanton, CA; Polla, Dennis L [Roseville, MN; Maghribi, Mariam N [Davis, CA; Hamilton, Julie [Tracy, CA; Humayun, Mark S [La Canada, CA; Weiland, James D [Valencia, CA

2012-01-28

An image is captured or otherwise converted into a signal in an artificial vision system. The signal is transmitted to the retina utilizing an implant. The implant consists of a polymer substrate made of a compliant material such as poly(dimethylsiloxane) or PDMS. The polymer substrate is conformable to the shape of the retina. Electrodes and conductive leads are embedded in the polymer substrate. The conductive leads and the electrodes transmit the signal representing the image to the cells in the retina. The signal representing the image stimulates cells in the retina.

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

PubMed Central

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-01-01

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. PMID:24606937

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

PubMed Central

Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R.

2015-01-01

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called “repeat-swap modeling” to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism. PMID:26388773

Nucleotide-induced conformational dynamics in ABC transporters from structure-based coarse grained modelling.

NASA Astrophysics Data System (ADS)

Flechsig, Holger

2016-02-01

ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter. Possible explanations are discussed in the light of currently debated transport scenarios of ABC transporters.

An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features

PubMed Central

Ruiz, Duncan D. A.; Norberto de Souza, Osmar

2015-01-01

Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward’s, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill biologically relevant information from MD trajectories, especially for docking purposes. PMID:26218832

An Effective Approach for Clustering InhA Molecular Dynamics Trajectory Using Substrate-Binding Cavity Features.

PubMed

De Paris, Renata; Quevedo, Christian V; Ruiz, Duncan D A; Norberto de Souza, Osmar

2015-01-01

Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward's, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill biologically relevant information from MD trajectories, especially for docking purposes.

Retuning Rieske-type Oxygenases to Expand Substrate Range

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

Mohammadi, Mahmood; Viger, Jean-François; Kumar, Pravindra

2012-09-17

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE{sub p4}, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE{sub LB400} by the double substitution T335A/F336M, and BphAE{sub RR41}, obtained by changing Asn{sup 338}, Ile{sup 341}, and Leu{sup 409} of BphAE{sub p4} to Gln{sup 338}, Val{sup 341}, and Phe{sup 409}, metabolize dibenzofuran two and three times faster than BphAE{sub LB400}, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE{sub LB400} showed thatmore » the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.« less

Handheld Chem/Biosensor Using Extreme Conformational Changes in Designed Binding Proteins to Enhance Surface Plasmon Resonance (SPR)

DTIC Science & Technology

2016-04-01

AFCEC-CX-TY-TR-2016-0007 HANDHELD CHEM/ BIOSENSOR USING EXTREME CONFORMATIONAL CHANGES IN DESIGNED BINDING PROTEINS TO ENHANCE SURFACE PLASMON...Include area code) 03/24/2016 Abstract 08/14/2015--03/31/2016 Handheld chem/ biosensor using extreme conformational changes in designed binding...Baltimore, Maryland on 17-21 April 2016. We propose the development of a highly sensitive handheld chem/ biosensor device using a novel class of engineered

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.

Common functionally important motions of the nucleotide-binding domain of Hsp70.

PubMed

Gołaś, Ewa I; Czaplewski, Cezary; Scheraga, Harold A; Liwo, Adam

2015-02-01

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate-binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (PDB 3C7N:B) by all-atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP- and ATP-unique classes, which reflect conformational trends that are unique to either the ADP- or ATP-bound states, respectively. "Mutual" class motions generally describe "in-plane" and/or "out-of-plane" (scissor-like) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The "unique" class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the "unique" type, regions of enhanced mobility can be identified; these are termed "hot spots," and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide-binding pocket was also found to influence the dynamics of the NBD significantly. © 2014 Wiley Periodicals, Inc.

Galilean field theories and conformal structure

NASA Astrophysics Data System (ADS)

Bagchi, Arjun; Chakrabortty, Joydeep; Mehra, Aditya

2018-04-01

We perform a detailed analysis of Galilean field theories, starting with free theories and then interacting theories. We consider non-relativistic versions of massless scalar and Dirac field theories before we go on to review our previous construction of Galilean Electrodynamics and Galilean Yang-Mills theory. We show that in all these cases, the field theories exhibit non-relativistic conformal structure (in appropriate dimensions). The surprising aspect of the analysis is that the non-relativistic conformal structure exhibited by these theories, unlike relativistic conformal invariance, becomes infinite dimensional even in spacetime dimensions greater than two. We then couple matter with Galilean gauge theories and show that there is a myriad of different sectors that arise in the non-relativistic limit from the parent relativistic theories. In every case, if the parent relativistic theory exhibited conformal invariance, we find an infinitely enhanced Galilean conformal invariance in the non-relativistic case. This leads us to suggest that infinite enhancement of symmetries in the non-relativistic limit is a generic feature of conformal field theories in any dimension.

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

PubMed Central

Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

1995-01-01

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

A pharmacophore model specific to active site of CYP1A2 with a novel molecular modeling explorer and CoMFA.

PubMed

Zhang, Tao; Wei, Dong-Qing; Chou, Kuo-Chen

2012-03-01

Comparative molecular field analysis (CoMFA) is a widely used 3D-QSAR method by which we can investigate the potential relation between biological activity of compounds and their structural features. In this study, a new application of this approach is presented by combining the molecular modeling with a new developed pharmacophore model specific to CYP1A2 active site. During constructing the model, we used the molecular dynamics simulation and molecular docking method to select the sensible binding conformations for 17 CYP1A2 substrates based on the experimental data. Subsequently, the results obtained via the alignment of binding conformations of substrates were projected onto the active- site residues, upon which a simple blueprint of active site was produced. It was validated by the experimental and computational results that the model did exhibit the high degree of rationality and provide useful insights into the substrate binding. It is anticipated that our approach can be extended to investigate the protein-ligand interactions for many other enzyme-catalyzed systems as well.

The role of enzyme distortion in the single displacement mechanism of family 19 chitinases

PubMed Central

Brameld, Ken A.; Goddard, William A.

1998-01-01

By using molecular dynamics simulations, we have examined the binding of a hexaNAG substrate and two potential hydrolysis intermediates (an oxazoline ion and an oxocarbenium ion) to a family 19 barley chitinase. We find the hexaNAG substrate binds with all sugars in a chair conformation, unlike the family 18 chitinase which causes substrate distortion. Glu 67 is in a position to protonate the anomeric oxygen linking sugar residues D and E whereas Asn 199 serves to hydrogen bond with the C2′ N-acetyl group of sugar D, thus preventing the formation of an oxazoline ion intermediate. In addition, Glu 89 is part of a flexible loop region allowing a conformational change to occur within the active site to bring the oxocarbenium ion intermediate and Glu 89 closer by 4–5 Å. A hydrolysis product with inversion of the anomeric configuration occurs because of nucleophilic attack by a water molecule that is coordinated by Glu 89 and Ser 120. Issues important for the design of inhibitors specific to family 19 chitinases over family 18 chitinases also are discussed. PMID:9539727

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations.

PubMed

Soares, Rosemberg O; Torres, Pedro H M; da Silva, Manuela L; Pascutti, Pedro G

2016-08-01

The active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pH-dependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25' and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding. Copyright © 2016 Elsevier Inc. All rights reserved.

Charged/Polar-Residue Scanning of the Hydrophobic Face of Transmembrane Domain 9 of the Yeast Glutathione Transporter, Hgt1p, Reveals a Conformationally Critical Region for Substrate Transport

PubMed Central

Thakur, Anil; Bachhawat, Anand K.

2015-01-01

Unraveling the mechanistic workings of membrane transporters has remained a challenging task. We describe a novel strategy that involves subjecting the residues of the hydrophobic face of a transmembrane helix to a charged/polar scanning mutagenesis. TMD9 of the yeast glutathione transporter, Hgt1p, has been identified as being important in substrate binding, and two residues, F523 and Q526, are expected to line the substrate translocation channel while the other face is hydrophobic. The hydrophobic face of TMD9 helix consists of residues A509, V513, L517, L520, I524, and I528, and these were mutated to lysine, glutamine, and glutamic acid. Among the 16 charged mutants created, six were nonfunctional, revealing a surprising tolerance of charged residues in the hydrophobic part of TM helices. Furthermore, the only position that did not tolerate any charged residue was I524, proximal to the substrate binding residues. However, P525, also proximal to the substrate binding residues, did tolerate charged/polar residues, suggesting that mere proximity to the substrate binding residues was not the only factor. The I524K/E/Q mutants expressed well and localized correctly despite lacking any glutathione uptake capability. Isolation of suppressors for all nonfunctional mutants yielded second-site suppressors only for I524K and I524Q, and suppressors for these mutations appeared at G202K/I and G202K/Q, respectively. G202 is in the hydrophilic loop between TMD3 and TMD4. The results suggest that I524 in the hydrophobic face interacts with this region and is also in a conformationally critical region for substrate translocation. PMID:25784163

Surface enhanced Raman optical activity of molecules on orientationally averaged substrates: theory of electromagnetic effects.

PubMed

Janesko, Benjamin G; Scuseria, Gustavo E

2006-09-28

We present a model for electromagnetic enhancements in surface enhanced Raman optical activity (SEROA) spectroscopy. The model extends previous treatments of SEROA to substrates, such as metal nanoparticles in solution, that are orientationally averaged with respect to the laboratory frame. Our theoretical treatment combines analytical expressions for unenhanced Raman optical activity with molecular polarizability tensors that are dressed by the substrate's electromagnetic enhancements. We evaluate enhancements from model substrates to determine preliminary scaling laws and selection rules for SEROA. We find that dipolar substrates enhance Raman optical activity (ROA) scattering less than Raman scattering. Evanescent gradient contributions to orientationally averaged ROA scale to first or higher orders in the gradient of the incident plane-wave field. These evanescent gradient contributions may be large for substrates with quadrupolar responses to the plane-wave field gradient. Some substrates may also show a ROA contribution that depends only on the molecular electric dipole-electric dipole polarizability. These conclusions are illustrated via numerical calculations of surface enhanced Raman and ROA spectra from (R)-(-)-bromochlorofluoromethane on various model substrates.

An Introduction to Atomic Layer Deposition with Thermal Applications

NASA Technical Reports Server (NTRS)

Dwivedi, Vivek H.

2015-01-01

Atomic Layer Deposition (ALD) is a cost effective nano-manufacturing technique that allows for the conformal coating of substrates with atomic control in a benign temperature and pressure environment. Through the introduction of paired precursor gases thin films can be deposited on a myriad of substrates ranging from glass, polymers, aerogels, and metals to high aspect ratio geometries. This talk will focus on the utilization of ALD for engineering applications.

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

PubMed

Hotta, Kinya; Ranganathan, Soumya; Liu, Ruchuan; Wu, Fei; Machiyama, Hiroaki; Gao, Rong; Hirata, Hiroaki; Soni, Neelesh; Ohe, Takashi; Hogue, Christopher W V; Madhusudhan, M S; Sawada, Yasuhiro

2014-04-01

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

PubMed

Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

2014-01-01

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

PubMed

Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

2004-10-01

In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

Direct Observation of Asphaltene Nanoparticles on Model Mineral Substrates.

PubMed

Raj, Gijo; Lesimple, Alain; Whelan, Jamie; Naumov, Panče

2017-06-27

The propensity for adherence to solid surfaces of asphaltenes, a complex solubility class of heteropolycyclic aromatic compounds from the heavy fraction of crude oil, has long been the root cause of scale deposition and remains an intractable problem in the petroleum industry. Although the adhesion is essential to understanding the process of asphaltene deposition, the relationship between the conformation of asphaltene molecules on mineral substrates and its impact on adhesion and mechanical properties of the deposits is not completely understood. To rationalize the primary processes in the process of organic scale deposition, here we use atomic force microscopy (AFM) to visualize the morphology of petroleum asphaltenes deposited on model mineral substrates. High imaging contrast was achieved by the differential adhesion of the tip between asphaltenes and the mineral substrate. While asphaltenes form smooth continuous films on all substrates at higher concentrations, they deposit as individual nanoparticles at lower concentrations. The size, shape, and spatial distribution of the nanoaggregates are strongly affected by the nature of the substrate; while uniformly distributed spherical particles are formed on highly polar and hydrophilic substrates (mica), irregular islands and thicker patches are observed with substrates of lower polarity (silica and calcite). Asphaltene nanoparticles flatten when adsorbed on highly oriented pyrolytic graphite due to π-π interactions with the polycyclic core. Force-distance profiles provide direct evidence of the conformational changes of asphaltene molecules on hydrophilic/hydrophobic substrates that result in dramatic changes in adhesion and mechanical properties of asphaltene deposits. Such an understanding of the nature of adhesion and mechanical properties tuned by surface properties, on the level of asphaltene nanoaggregates, would contribute to the design of efficient asphaltene inhibitors for preventing asphaltene fouling on targeted surfaces. Unlike flat surfaces, the AFM phase contrast images of defected calcite surfaces show that asphaltenes form continuous deposits to fill the recesses, and this process could trigger the onset for asphaltene deposition.

Fabricating nanowire devices on diverse substrates by simple transfer-printing methods.

PubMed

Lee, Chi Hwan; Kim, Dong Rip; Zheng, Xiaolin

2010-06-01

The fabrication of nanowire (NW) devices on diverse substrates is necessary for applications such as flexible electronics, conformable sensors, and transparent solar cells. Although NWs have been fabricated on plastic and glass by lithographic methods, the choice of device substrates is severely limited by the lithographic process temperature and substrate properties. Here we report three new transfer-printing methods for fabricating NW devices on diverse substrates including polydimethylsiloxane, Petri dishes, Kapton tapes, thermal release tapes, and many types of adhesive tapes. These transfer-printing methods rely on the differences in adhesion to transfer NWs, metal films, and devices from weakly adhesive donor substrates to more strongly adhesive receiver substrates. Electrical characterization of fabricated NW devices shows that reliable ohmic contacts are formed between NWs and electrodes. Moreover, we demonstrated that Si NW devices fabricated by the transfer-printing methods are robust piezoresistive stress sensors and temperature sensors with reliable performance.

Implantable biomedical devices on bioresorbable substrates

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

Rogers, John A; Kim, Dae-Hyeong; Omenetto, Fiorenzo

Provided herein are implantable biomedical devices, methods of administering implantable biomedical devices, methods of making implantable biomedical devices, and methods of using implantable biomedical devices to actuate a target tissue or sense a parameter associated with the target tissue in a biological environment. Each implantable biomedical device comprises a bioresorbable substrate, an electronic device having a plurality of inorganic semiconductor components supported by the bioresorbable substrate, and a barrier layer encapsulating at least a portion of the inorganic semiconductor components. Upon contact with a biological environment the bioresorbable substrate is at least partially resorbed, thereby establishing conformal contact between themore » implantable biomedical device and the target tissue in the biological environment.« less

Extraordinary optical transmission in nanopatterned ultrathin metal films without holes

DOE PAGES

Peer, Akshit; Biswas, Rana

2016-02-01

In this study, we experimentally and theoretically demonstrate that a continuous gold film on a periodically textured substrate exhibits extraordinary optical transmission, even though no holes were etched in the film. Our film synthesis started by nanoimprinting a periodic array of nanocups with a period of ~750 nm on a polystyrene film over a glass substrate. A thin non-conformal gold film was sputter-deposited on the polystyrene by angle-directed deposition. The gold film was continuous with spatial thickness variation, the film being thinnest at the bottom of the nanocup. Measurements revealed an extraordinary transmission peak at a wavelength just smaller thanmore » the period, with an enhancement of ~2.5 compared to the classically expected value. Scattering matrix simulations model well the transmission and reflectance measurements when an ultrathin gold layer (~5 nm), smaller than the skin depth is retained at the bottom of the nanocups. Electric field intensities are enhanced by >100 within the nanocup, and ~40 in the ultrathin gold layer causing transmission through it. We show a wavelength red-shift of ~30 nm in the extraordinary transmission peak when the nanocups are coated with a thin film of a few nanometers, which can be utilized for biosensing. The continuous corrugated metal films are far simpler structures to observe extraordinary transmission, circumventing the difficult process of etching the metal film. Such continuous metal films with ultrathin regions are simple platforms for non-linear optics, plasmonics, and biological and chemical sensing.« less

A Twist on Facial Selectivity of Hydride Reductions of Cyclic Ketones: Twist-Boat Conformers in Cyclohexanone, Piperidone, and Tropinone Reactions

PubMed Central

2015-01-01

The role of twist-boat conformers of cyclohexanones in hydride reductions was explored. The hydride reductions of a cis-2,6-disubstituted N-acylpiperidone, an N-acyltropinone, and tert-butylcyclohexanone by lithium aluminum hydride and by a bulky borohydride reagent were investigated computationally and compared to experiment. Our results indicate that in certain cases, factors such as substrate conformation, nucleophile bulkiness, and remote steric features can affect stereoselectivity in ways that are difficult to predict by the general Felkin–Anh model. In particular, we have calculated that a twist-boat conformation is relevant to the reactivity and facial selectivity of hydride reduction of cis-2,6-disubstituted N-acylpiperidones with a small hydride reagent (LiAlH4) but not with a bulky hydride (lithium triisopropylborohydride). PMID:25372509

Flexibility, Diversity, and Cooperativity: Pillars of Enzyme Catalysis

PubMed Central

Hammes, Gordon G.; Benkovic, Stephen J.; Hammes-Schiffer, Sharon

2011-01-01

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, NMR, and single molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model. PMID:22029278

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.

The ABC protein turned chloride channel whose failure causes cystic fibrosis

NASA Astrophysics Data System (ADS)

Gadsby, David C.; Vergani, Paola; Csanády, László

2006-03-01

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

PubMed Central

Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

2015-01-01

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

Optogenetic Control of Cells and Circuits

PubMed Central

Miesenböck, Gero

2013-01-01

The absorption of light by bound or diffusible chromophores causes conformational rearrangements in natural and artificial photoreceptor proteins. These rearrangements are coupled to the opening or closing of ion transport pathways, the association or dissociation of binding partners, the enhancement or suppression of catalytic activity, or the transcription or repression of genetic information. Illumination of cells, tissues, or organisms engineered genetically to express photoreceptor proteins can thus be used to perturb biochemical and electrical signaling with exquisite cellular and molecular specificity. First demonstrated in 2002, this principle of optogenetic control has had a profound impact on neuroscience, where it provides a direct and stringent means of probing the organization of neural circuits and of identifying the neural substrates of behavior. The impact of optogenetic control is also beginning to be felt in other areas of cell and organismal biology. PMID:21819234

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

PubMed Central

Huang, Rui; Ripstein, Zev A.; Augustyniak, Rafal; Lazniewski, Michal; Ginalski, Krzysztof; Kay, Lewis E.; Rubinstein, John L.

2016-01-01

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded. PMID:27402735

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*

PubMed Central

Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.

2015-01-01

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

New approach for producing chemical templates over large area by Molecular Transfer Printing

NASA Astrophysics Data System (ADS)

Inoue, Takejiro; Janes, Dustin; Ren, Jiaxing; Willson, Grant; Ellison, Christopher; Nealey, Paul

2014-03-01

Fabrication of well-defined chemically patterned surfaces is crucially important to the development of next generation microprocessors, hard disk memory devices, photonic/plasmonic devices, separation membranes, and biological microarrays. One promising patterning method in these fields is Molecular Transfer Printing (MTP), which replicates chemical patterns with feature dimensions of the order of 10nm utilizing a master template defined by the microphase separated domains of a block copolymer thin film. The total transfer printing area achievable by MTP has so far been limited by the contact area between two rigid substrates. Therefore, strategies to make conformal contact between substrates could be practically useful because a single lithographically-defined starting pattern could be used to fabricate many replicates by a low-cost process. Here we show a new approach that utilizes a chemically deposited SiN layer and a liquid conformal layer to enable transfer printing of chemical patterns upon thermal annealing over large, continuous areas. We anticipate that our process could be integrated into Step and Flash Imprint Lithography (SFIL) tools to achieve conformal layer thicknesses thin and uniform enough to permit pattern transfer through a dry-etch protocol.

DapF stabilizes the substrate-favoring conformation of RppH to stimulate its RNA-pyrophosphohydrolase activity in Escherichia coli.

PubMed

Wang, Qiang; Zhang, Delin; Guan, Zeyuan; Li, Dongqin; Pei, Kai; Liu, Jian; Zou, Tingting; Yin, Ping

2018-06-21

mRNA decay is an important strategy by which bacteria can rapidly adapt to their ever-changing surroundings. The 5'-terminus state of mRNA determines the velocity of decay of many types of RNA. In Escherichia coli, RNA pyrophosphohydrolase (RppH) is responsible for the removal of the 5'-terminal triphosphate from hundreds of mRNAs and triggers its rapid degradation by ribonucleases. A diaminopimelate epimerase, DapF, can directly interact with RppH and stimulate its hydrolysis activity in vivo and in vitro. However, the molecular mechanism remains to be elucidated. Here, we determined the complex structure of DapF-RppH as a heterotetramer in a 2:2 molar ratio. DapF-bound RppH exhibits an RNA-favorable conformation similar to the RNA-bound state, suggesting that association with DapF promotes and stabilizes RppH in a conformation that facilitates substrate RNA binding and thus stimulates the activity of RppH. To our knowledge, this is the first published structure of an RNA-pyrophosphohydrolysis complex in bacteria. Our study provides a framework for further investigation of the potential regulators involved in the RNA-pyrophosphohydrolysis process in prokaryotes.

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.

Combination of inverted pyramidal nanovoid with silver nanoparticles to obtain further enhancement and its detection for ricin

NASA Astrophysics Data System (ADS)

Wang, Meng; Wang, Bin; Wu, Shixuan; Guo, Tingke; Li, Haoyu; Guo, Zhaoqing; Wu, Junhua; Jia, Peiyuan; Wang, Yuxia; Xu, Xiaoxuan; Wang, Yufang; Zhang, Cunzhou

2015-02-01

We have obtained the surface-enhanced Raman scattering substrate by depositing silver nanoparticles on the surface of the inverted pyramidal nanovoid in order to improve the enhance effects. Experimental results showed that the combined substrate exhibited greater enhancement than the nanovoid substrate or nanoparticles. In order to test the SERS activity of the combined substrates, Rh6G and ricin toxin were used as Raman probes. Finite element method was employed to simulate electric field and induced charge distribution of the substrates, which have been used to explore the interaction between nanoparticles and nanovoid as well as mechanism of the great enhancement.

Transferred nuclear Overhauser enhancement experiments show that the monoclonal antibody strep 9 selects a local minimum conformation of a Streptococcus group A trisaccharide-hapten.

PubMed

Weimar, T; Harris, S L; Pitner, J B; Bock, K; Pinto, B M

1995-10-17

Transferred nuclear Overhauser enhancement (TRNOE) experiments have been performed to investigate the bound conformation of the trisaccharide repeating unit of the Streptococcus Group A cell-wall polysaccharide. Thus, the conformations of propyl 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-O-(alpha-L-rhamnopyran osyl)- alpha-L-rhamnopyranoside [C(A')B] (1) as a free ligand and when complexed to the monoclonal antibody Strep 9 were examined. Improved insights about the conformational preferences of the glycosidic linkages of the trisaccharide ligand showed that the free ligand populates various conformations in aqueous solution, thus displaying relatively flexible behavior. The NOE HNAc-H2A', which was not detected in previous work, accounts for a conformation at the beta-(1-->3) linkage with a phi angle of approximately 180 degrees. Observed TRNOEs for the complex are weak, and their analysis was further complicated by spin diffusion. With the use of transferred rotating-frame Overhauser enhancement (TRROE) experiments, the amount of spin diffusion was assessed experimentally, proving that all of the observed long-range TRNOEs arose through spin diffusion. Four interglycosidic distances, derived from the remaining TRNOEs and TRROEs, together with repulsive constraints, derived from the absence of TRROE effects, were used as input parameters in simulated annealing and molecular mechanics calculations to determine the bound conformation of the trisaccharide. Complexation by the antibody results in the selection of one defined conformation of the carbohydrate hapten. This bound conformation, which is a local energy minimum on the energy maps calculated for the trisaccharide ligand, shows only a change from a +gauche to a -gauche orientation at the psi angle of the alpha-(1-->2) linkage when compared to the global minimum conformation. The results infer that the bound conformation of the Streptococcus Group A cell-wall polysaccharide is different from its previously proposed solution structure (Kreis et al., 1995).

pH-directed self-assembling helical peptide conformation

USDA-ARS?s Scientific Manuscript database

The beta-sheet and alpha-helix peptide conformation are two of the most fundamentally ordered secondary structures found in proteins and peptides. They also give rise to self-assembling motifs that form macromolecular channels and nanostructures. Through design these conformations can yield enhance...

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

Catalysis by Orotidine 5′-Monophosphate Decarboxylase: Effect of 5-Fluoro and 4′-Substituents on the Decarboxylation of Two-Part Substrates†

PubMed Central

Goryanova, Bogdana; Spong, Krisztina; Amyes, Tina L.; Richard, John P.

2013-01-01

The syntheses of two novel truncated analogs of the natural substrate orotidine 5′-monophosphate (OMP) for orotidine 5′-monophosphate decarboxylase (OMPDC) with enhanced reactivity towards decarboxylation are reported: 1-(β-D-erythrofuranosyl)-5-fluoroorotic acid (FEO) and 5′-deoxy-5-fluoroorotidine (5′-dFO). A comparison of the second-order rate constants for the OMPDC-catalyzed decarboxylations of FEO (10 M−1 s−1) and 1-(β-D-erythrofuranosyl)orotic acid (EO, 0.026 M−1 s−1) shows that the vinyl carbanion-like transition state is stabilized by 3.5 kcal/mol by interactions with the 5-F substituent of FEO. The OMPDC-catalyzed decarboxylations of FEO and EO are both activated by exogenous phosphite dianion (HPO32−), but the 5-F substituent results in only a 0.8 kcal stabilization of the transition state for the phosphite-activated reaction of FEO. This provides strong evidence that the phosphite-activated OMPDC-catalyzed reaction of FEO is not limited by the chemical step of decarboxylation of the enzyme-bound substrate. Evidence is presented that there is a change in rate-limiting step from the chemical step of decarboxylation for the phosphite-activated reaction of EO, to closure of the phosphate gripper loop and an enzyme conformational change at the ternary E·FEO·HPO32− complex for the reaction of FEO. The 4′-CH3 and 4′-CH2OH groups of 5′-dFO and orotidine, respectively, result in identical destabilizations of the transition state for the unactivated decarboxylation of 2.9 kcal/mol. By contrast, the 4′-CH3 group of 5′-dFO and the 4′-CH2OH group of orotidine result in very different 4.7 and 8.3 kcal/mol destabilizations of the transition state for the phosphite-activated decarboxylation. Here, the destabilizing effect of the 4′-CH3 substituent at 5′-dFO is masked by the rate-limiting conformational change that depresses the third-order rate constant for the phosphite-activated reaction of the parent substrate FEO. PMID:23276261

Conformational flexibility of human casein kinase catalytic subunit explored by metadynamics.

PubMed

Gouron, Aurélie; Milet, Anne; Jamet, Helene

2014-03-04

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Conformational Sampling and Nucleotide-Dependent Transitions of the GroEL Subunit Probed by Unbiased Molecular Dynamics Simulations

PubMed Central

Skjaerven, Lars; Grant, Barry; Muga, Arturo; Teigen, Knut; McCammon, J. Andrew; Reuter, Nathalie; Martinez, Aurora

2011-01-01

GroEL is an ATP dependent molecular chaperone that promotes the folding of a large number of substrate proteins in E. coli. Large-scale conformational transitions occurring during the reaction cycle have been characterized from extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in the allosteric response to ATP and the nucleotide-driven reaction cycle are not completely established. Here we describe extensive (in total long) unbiased molecular dynamics (MD) simulations that probe the response of GroEL subunits to ATP binding. We observe nucleotide dependent conformational transitions, and show with multiple 100 ns long simulations that the ligand-induced shift in the conformational populations are intrinsically coded in the structure-dynamics relationship of the protein subunit. Thus, these simulations reveal a stabilization of the equatorial domain upon nucleotide binding and a concomitant “opening” of the subunit, which reaches a conformation close to that observed in the crystal structure of the subunits within the ADP-bound oligomer. Moreover, we identify changes in a set of unique intrasubunit interactions potentially important for the conformational transition. PMID:21423709

A pH-responsive interface derived from resilin-mimetic protein Rec1-resilin.

PubMed

Truong, My Y; Dutta, Naba K; Choudhury, Namita R; Kim, Misook; Elvin, Christopher M; Hill, Anita J; Thierry, Benjamin; Vasilev, Krasimir

2010-05-01

In this investigation, for the first time we report the effects of pH on the molecular orientation, packing density, structural properties, adsorption characteristics and viscoelastic behaviour of resilin-mimetic protein rec1-resilin at the solid-liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) spectroscopy. QCM-D and SPR data confirm that the binding ability of rec1-resilin on a substrate is strongly pH-dependent the protein packing density on a gold surface is calculated to be 4.45 x 10(13) per cm(2) at the isoelectric point (IEP approximately 4.9), 8.79 x 10(11) per cm(2) at pH 2 and 9.90 x 10(11) per cm(2) at pH 12, respectively. Our findings based on the thickness, dissipation and viscoelasticity of the rec1-resilin adlayer also indicate that it is adsorbed onto the gold substrate with different orientation depending on pH, such as back-on adsorption at acidic pH of 2, compact end-on bilayer adsorption at the IEP and side-on at high alkaline pH of 12. When rec1-resilin is 'pinned' to the substrate at IEP and subsequently exposed to an electrolyte solution adjusted to different pH, it switches from a compact globular conformation of the bio-macromolecule at the IEP to a coil conformation at pH between IEP to IED (IED = pKa value of tyrosine amino acid residue) and an extended coil conformation at pH > IED. This transformation from globule to coil to extended coil conformation is kinetically fast, robust and completely reversible. Such responsive surfaces created using 'smart' biomimetic rec1-resilin have the potential to find applications in many areas including biotechnology, medicine, sensors, controlled drug delivery systems and engineering. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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.

The conformational dynamics of the mitochondrial Hsp70 chaperone.

PubMed

Mapa, Koyeli; Sikor, Martin; Kudryavtsev, Volodymyr; Waegemann, Karin; Kalinin, Stanislav; Seidel, Claus A M; Neupert, Walter; Lamb, Don C; Mokranjac, Dejana

2010-04-09

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments. 2010 Elsevier Inc. All rights reserved.

Conformational adaptation and manipulation of manganese tetra(4-pyridyl)porphyrin molecules on Cu(111)

NASA Astrophysics Data System (ADS)

Chen, Xianwen; Lei, Shulai; Lotze, Christian; Czekelius, Constantin; Paulus, Beate; Franke, Katharina J.

2017-03-01

Porphyrins are highly flexible molecules and well known to adapt to their local environment via conformational changes. We studied the self-assembly of manganese meso-tetra(4-pyridyl)porphyrin (Mn-TPyP) molecules on a Cu(111) surface by low temperature scanning tunneling microscopy (STM) and atomic force microscopy (ATM). We observe molecular chains along the ⟨1 1 ¯ 0 ⟩ direction of the substrate. Within these chains, we identify two molecular conformations, which differ by the orientation of the upward bending of the macrocycle. Using density functional theory, we show that this saddle shape is a consequence of the rotation and inclination of the pyridyl groups towards Cu adatoms, which stabilize the metal-organic chains. The molecular conformations obey a strict alternation, reflecting the mutual enforcement of conformational adaptation in densely packed structures. Tunneling electrons from the STM tip can induce changes in the orientation of the pyridyl endgroups. The switching behaviour varies with the different adsorption configurations.

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

PubMed

Wasserman, Michael R; Alejo, Jose L; Altman, Roger B; Blanchard, Scott C

2016-04-01

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Multi-perspective smFRET reveals rate-determining late intermediates of ribosomal translocation

PubMed Central

Wasserman, Michael R.; Alejo, Jose L.; Altman, Roger B.; Blanchard, Scott C.

2016-01-01

Directional translocation of the ribosome through the messenger RNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of transfer and messenger RNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we have tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations reveal direct evidence of structurally and kinetically distinct, late intermediates during substrate movement, whose resolution is rate-determining to the translocation mechanism. These steps involve intra-molecular events within the EFG(GDP)-bound ribosome, including exaggerated, reversible fluctuations of the small subunit head domain, which ultimately facilitate peptidyl-tRNA’s movement into its final post-translocation position. PMID:26926435

Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE

PubMed Central

Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G.; Zheng, Hongjin; Gonen, Tamir

2014-01-01

The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins. PMID:25088546

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

Characterization of Next Generation Commercial Surface Enhanced Raman Scattering Substrates with a 633- and 785-nm System

DTIC Science & Technology

2013-04-01

Characterization of Next Generation Commercial Surface Enhanced Raman Scattering Substrates with a 633- and 785-nm System by Mikella E...Surface Enhanced Raman Scattering Substrates with a 633- and 785-nm System Mikella E. Farrell, Dimitra N. Stratis-Cullum, and Paul M. Pellegrino...DATES COVERED (From - To) 4. TITLE AND SUBTITLE Characterization of Next Generation Commercial Surface Enhanced Raman Scattering Substrates with a

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.

Superstrong encapsulated monolayer graphene by the modified anodic bonding

NASA Astrophysics Data System (ADS)

Jung, Wonsuk; Yoon, Taeshik; Choi, Jongho; Kim, Soohyun; Kim, Yong Hyup; Kim, Taek-Soo; Han, Chang-Soo

2013-12-01

We report a superstrong adhesive of monolayer graphene by modified anodic bonding. In this bonding, graphene plays the role of a superstrong and ultra-thin adhesive between SiO2 and glass substrates. As a result, monolayer graphene presented a strong adhesion energy of 1.4 J m-2 about 310% that of van der Waals bonding (0.45 J m-2) to SiO2 and glass substrates. This flexible solid state graphene adhesive can tremendously decrease the adhesive thickness from about several tens of μm to 0.34 nm for epoxy or glue at the desired bonding area. As plausible causes of this superstrong adhesion, we suggest conformal contact with the rough surface of substrates and generation of C-O chemical bonding between graphene and the substrate due to the bonding process, and characterized these properties using optical microscopy, atomic force microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy.We report a superstrong adhesive of monolayer graphene by modified anodic bonding. In this bonding, graphene plays the role of a superstrong and ultra-thin adhesive between SiO2 and glass substrates. As a result, monolayer graphene presented a strong adhesion energy of 1.4 J m-2 about 310% that of van der Waals bonding (0.45 J m-2) to SiO2 and glass substrates. This flexible solid state graphene adhesive can tremendously decrease the adhesive thickness from about several tens of μm to 0.34 nm for epoxy or glue at the desired bonding area. As plausible causes of this superstrong adhesion, we suggest conformal contact with the rough surface of substrates and generation of C-O chemical bonding between graphene and the substrate due to the bonding process, and characterized these properties using optical microscopy, atomic force microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03822j

Control by substrate of the cytochrome p450-dependent redox machinery: mechanistic insights.

PubMed

Hlavica, Peter

2007-08-01

Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading toxic pollutants. Finally, availability of P450 nanobiochips for high-throughput screening of substrate libraries might expedite drug development.

Co-existence of Distinct Prion Types Enables Conformational Evolution of Human PrPSc by Competitive Selection*

PubMed Central

Haldiman, Tracy; Kim, Chae; Cohen, Yvonne; Chen, Wei; Blevins, Janis; Qing, Liuting; Cohen, Mark L.; Langeveld, Jan; Telling, Glenn C.; Kong, Qingzhong; Safar, Jiri G.

2013-01-01

The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrPSc). The molecular mechanism responsible for the adaptation, mutation, and evolution of prions observed in cloned cells and upon crossing the species barrier remains unsolved. Using biophysical techniques and conformation-dependent immunoassays in tandem, we isolated two distinct populations of PrPSc particles with different conformational stabilities and aggregate sizes, which frequently co-exist in the most common human prion disease, sporadic Creutzfeldt-Jakob disease. The protein misfolding cyclic amplification replicates each of the PrPSc particle types independently and leads to the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrPC substrate, the dominant PrPSc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively, the data show that sporadic Creutzfeldt-Jakob disease PrPSc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions, whose adaptation and evolution are governed by the selection of progressively less stable, faster replicating PrPSc conformers. PMID:23974118

Dissociation of recombinant prion autocatalysis from infectivity.

PubMed

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Injectable Reactive Biocomposites For Bone Healing In Critical-Size Rabbit Calvarial Defects

DTIC Science & Technology

2012-03-29

defects (i.e. be conformable), provide temporary protection to the brain until the bone heals, and enhance tissue regeneration with the delivery of...temporary protection to the brain until the bone heals, and enhance tissue regeneration with the delivery of biologics. In this study, we evaluated the...complex defects (i.e. be conformable), harden to provide temporary protection until tissue remodels (i.e. be settable), and enhance tissue regeneration

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.

Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

PubMed

Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

2017-10-01

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

Influence of natural organic matter (NOM) and synthetic polyelectrolytes on colloidal behavior of metal oxide nanoparticles

NASA Astrophysics Data System (ADS)

Ghosh, Saikat

The colloidal behavior of engineered nanomaterials exposed in an aquatic environment may significantly influence their bioavailability as well as toxicity to different species. Natural organic matter (NOM) is one of the major colloidal materials ubiquitous in the environment with significant structural heterogeneity. Therefore, role of NOM molecules on environmental fate of these engineered NPs needs to be addressed. Colloidal behavior of aluminum (Al2O 3) and magnetic iron oxide (gammaFe2O3) NPs was studied in the presence of structurally different HAs and synthetic polyacrylic acids (PAAs). The conformation behavior of the adsorobed NOM/polyelectrolyte under specific solution conditions were determined with dynamic light scattering, atomic force microscopy measurements. Al2O3 NPs followed the classical DLVO model of colloidal behavior in their pristine state. However, a significant deviation from the classical DLVO model was observed when these NPs were coated with structurally different HAs. Low polar, high molecular weight HA fractions showed much stronger stabilization against Ca2+ induced aggregation. Previously, we observed that these low polar, high molecular weight fractions strongly destabilized the NP suspension when added in a small quantity. A significant transformation in suspension stability was observed possibly due to steric effect of these adsorbed HAs. The colloidal behavior of PAA/NOM coated ferrimagnetic gammaFe 2O3 NPs were investigated. Pure gammaFe2O 3 NPs were extremely unstable in aqueous solution but a significant enhancement in colloidal stability was observed after coating with polyelectrolytes/NOM. The steric as well as electrostatic stabilization introduced by the polyelectrolyte coating strongly dictated the colloidal stability. The alteration of electrosteric stabilization mechanisms by pH-induced conformation change profoundly influences the colloidal stability. Atomic force microscopy (AFM) study revealed a highly stretched conformation of the HA molecular chains adsorbed on gammaFe 2O3 NP surface with increasing pH from 5 to 9 which enhanced the colloidal stability trough long range electrosteric stabilization. The depletion of the polyelectrolytes during dilution of the suspension in the acidic solution conditions and in the presence of Na+ or Ca 2+ decreased the colloidal stability. The conformation of the polyelectrolytes adsorbed on the NP surface altered significantly as a function of substrate surface charge as viewed from the AFM imaging.

Direct fabrication of 3D graphene on nanoporous anodic alumina by plasma-enhanced chemical vapor deposition

PubMed Central

Zhan, Hualin; Garrett, David J.; Apollo, Nicholas V.; Ganesan, Kumaravelu; Lau, Desmond; Prawer, Steven; Cervenka, Jiri

2016-01-01

High surface area electrode materials are of interest for a wide range of potential applications such as super-capacitors and electrochemical cells. This paper describes a fabrication method of three-dimensional (3D) graphene conformally coated on nanoporous insulating substrate with uniform nanopore size. 3D graphene films were formed by controlled graphitization of diamond-like amorphous carbon precursor films, deposited by plasma-enhanced chemical vapour deposition (PECVD). Plasma-assisted graphitization was found to produce better quality graphene than a simple thermal graphitization process. The resulting 3D graphene/amorphous carbon/alumina structure has a very high surface area, good electrical conductivity and exhibits excellent chemically stability, providing a good material platform for electrochemical applications. Consequently very large electrochemical capacitance values, as high as 2.1 mF for a sample of 10 mm3, were achieved. The electrochemical capacitance of the material exhibits a dependence on bias voltage, a phenomenon observed by other groups when studying graphene quantum capacitance. The plasma-assisted graphitization, which dominates the graphitization process, is analyzed and discussed in detail. PMID:26805546

Direct fabrication of 3D graphene on nanoporous anodic alumina by plasma-enhanced chemical vapor deposition.

PubMed

Zhan, Hualin; Garrett, David J; Apollo, Nicholas V; Ganesan, Kumaravelu; Lau, Desmond; Prawer, Steven; Cervenka, Jiri

2016-01-25

High surface area electrode materials are of interest for a wide range of potential applications such as super-capacitors and electrochemical cells. This paper describes a fabrication method of three-dimensional (3D) graphene conformally coated on nanoporous insulating substrate with uniform nanopore size. 3D graphene films were formed by controlled graphitization of diamond-like amorphous carbon precursor films, deposited by plasma-enhanced chemical vapour deposition (PECVD). Plasma-assisted graphitization was found to produce better quality graphene than a simple thermal graphitization process. The resulting 3D graphene/amorphous carbon/alumina structure has a very high surface area, good electrical conductivity and exhibits excellent chemically stability, providing a good material platform for electrochemical applications. Consequently very large electrochemical capacitance values, as high as 2.1 mF for a sample of 10 mm(3), were achieved. The electrochemical capacitance of the material exhibits a dependence on bias voltage, a phenomenon observed by other groups when studying graphene quantum capacitance. The plasma-assisted graphitization, which dominates the graphitization process, is analyzed and discussed in detail.

Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

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

Watanabe, N.; Clay, M.D.; Belkum, M.J.van

2009-05-26

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) andmore » N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.« less

Direct synthesis and characterization of optically transparent conformal zinc oxide nanocrystalline thin films by rapid thermal plasma CVD

NASA Astrophysics Data System (ADS)

Pedersen, Joachim D.; Esposito, Heather J.; Teh, Kwok Siong

2011-10-01

We report a rapid, self-catalyzed, solid precursor-based thermal plasma chemical vapor deposition process for depositing a conformal, nonporous, and optically transparent nanocrystalline ZnO thin film at 130 Torr (0.17 atm). Pure solid zinc is inductively heated and melted, followed by ionization by thermal induction argon/oxygen plasma to produce conformal, nonporous nanocrystalline ZnO films at a growth rate of up to 50 nm/min on amorphous and crystalline substrates including Si (100), fused quartz, glass, muscovite, c- and a-plane sapphire (Al2O3), gold, titanium, and polyimide. X-ray diffraction indicates the grains of as-deposited ZnO to be highly textured, with the fastest growth occurring along the c-axis. The individual grains are observed to be faceted by (103) planes which are the slowest growth planes. ZnO nanocrystalline films of nominal thicknesses of 200 nm are deposited at substrate temperatures of 330°C and 160°C on metal/ceramic substrates and polymer substrates, respectively. In addition, 20-nm- and 200-nm-thick films are also deposited on quartz substrates for optical characterization. At optical spectra above 375 nm, the measured optical transmittance of a 200-nm-thick ZnO film is greater than 80%, while that of a 20-nm-thick film is close to 100%. For a 200-nm-thick ZnO film with an average grain size of 100 nm, a four-point probe measurement shows electrical conductivity of up to 910 S/m. Annealing of 200-nm-thick ZnO films in 300 sccm pure argon at temperatures ranging from 750°C to 950°C (at homologous temperatures between 0.46 and 0.54) alters the textures and morphologies of the thin film. Based on scanning electron microscope images, higher annealing temperatures appear to restructure the ZnO nanocrystalline films to form nanorods of ZnO due to a combination of grain boundary diffusion and bulk diffusion. PACS: films and coatings, 81.15.-z; nanocrystalline materials, 81.07.Bc; II-VI semiconductors, 81.05.Dz.

Apo adenylate kinase encodes its holo form: a principal component and varimax analysis.

PubMed

Cukier, Robert I

2009-02-12

Adenylate kinase undergoes large-scale motions of its LID and AMP-binding (AMPbd) domains when its apo, open form closes over its substrates, AMP and Mg2+-ATP. It may be an example of an enzyme that provides an ensemble of conformations in its apo state from which its substrates can select and bind to produce catalytically competent conformations. In this work, the fluctuations of the enzyme apo Escherichia coli adenylate kinase (AKE) are obtained with molecular dynamics. The resulting trajectory is analyzed with principal component analysis (PCA) that decomposes the atom motions into orthogonal modes ordered by their decreasing contributions to the total protein fluctuation. In apo AKE, a small set of the PCA modes describes the bulk of the fluctuations. Identification of the atom motions that are important contributors to these modes is improved with the use of a varimax rotation method that rotates the PCA modes to a new mode set that concentrates the atom contributions to a smaller set of atoms in these new modes. In this way, the nature of the important motions of the LID and AMPbd domains are clarified. The dominant PCA modes are used to investigate if apo AKE can fluctuate to conformations that are holo-like, even though the apo trajectory is mainly confined to a region around the initial apo structure. This is accomplished by expressing the difference between the protein coordinates, obtained from the holo and apo crystal structures, using as a basis the PCA modes from the apo AKE trajectory. The coherent motion described by a small set of the apo PCA modes is shown to be able to produce protein conformations that are quite similar to the holo conformation of the protein. In this sense, apo AKE does encode in its fluctuations information about holo-like conformations.

Structural insight into the substrate- and dioxygen-binding manner in the catalytic cycle of rieske nonheme iron oxygenase system, carbazole 1,9a-dioxygenase.

PubMed

Ashikawa, Yuji; Fujimoto, Zui; Usami, Yusuke; Inoue, Kengo; Noguchi, Haruko; Yamane, Hisakazu; Nojiri, Hideaki

2012-06-24

Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex. In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding. The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O-O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Flexible Charged Macromolecules on Mixed Fluid Lipid Membranes: Theory and Monte Carlo Simulations

PubMed Central

Tzlil, Shelly; Ben-Shaul, Avinoam

2005-01-01

Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein. PMID:16126828

Structural and mechanistic insights into Mps1 kinase activation.

PubMed

Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

2009-08-01

Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-A-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices EF and F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

Synthesis of Graphene Based Membranes: Effect of Substrate Surface Properties on Monolayer Graphene Transfer.

PubMed

Kafiah, Feras; Khan, Zafarullah; Ibrahim, Ahmed; Atieh, Muataz; Laoui, Tahar

2017-01-21

In this work, we report the transfer of graphene onto eight commercial microfiltration substrates having different pore sizes and surface characteristics. Monolayer graphene grown on copper by the chemical vapor deposition (CVD) process was transferred by the pressing method over the target substrates, followed by wet etching of copper to obtain monolayer graphene/polymer membranes. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and contact angle (CA) measurements were carried out to explore the graphene layer transferability. Three factors, namely, the substrate roughness, its pore size, and its surface wetting (degree of hydrophobicity) are found to affect the conformality and coverage of the transferred graphene monolayer on the substrate surface. A good quality graphene transfer is achieved on the substrate with the following characteristics; being hydrophobic (CA > 90°), having small pore size, and low surface roughness, with a CA to RMS (root mean square) ratio higher than 2.7°/nm.

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

PubMed Central

Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

2015-01-01

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters. PMID:26238402

High performance and reusable SERS substrates using Ag/ZnO heterostructure on periodic silicon nanotube substrate

NASA Astrophysics Data System (ADS)

Lai, Yi-Chen; Ho, Hsin-Chia; Shih, Bo-Wei; Tsai, Feng-Yu; Hsueh, Chun-Hway

2018-05-01

Surface-enhanced Raman scattering (SERS) substrate with a higher surface area, enhanced light harvesting, multiple hot spots and strong electromagnetic field enhancements would exhibit enhanced Raman signals. Herein, the Ag nanoparticle/ZnO nanowire heterostructure decorated periodic silicon nanotube (Ag@ZnO@SiNT) substrate was proposed and fabricated. The proposed structure employed as SERS-active substrate was examined, and the results showed both the high performance in terms of high sensitivity and good reproducibility. Furthermore, the Ag@ZnO@SiNT substrate demonstrated the self-cleaning performance through the photocatalytic degradation of probed molecules upon UV-irradiation. The results showed that the proposed nanostructure had high performance, good reproducibility and reusability, and it is a promising SERS-active substrate for molecular sensing and cleaning.

The characterization of photographic materials as substrates for surface enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Vaughan, J.; Hortin, N.; Christie, S.; Kvasnik, F.; Scully, P. J.

2005-06-01

In this study, five types of photographic materials were obtained from commercial sources and characterized for use as substrates for surface enhanced Raman spectroscopy. The substrates are photographic emulsions coated on glass or paper support. The emulsions were developed to maximize the amount of metallic silver aggregated into clusters. The test analyte, Cresyl Violet, was deposited directly onto the substrate surface. The permeable nature of the supporting gelatin matrix enables the interaction between the target analyte and the solid silver clusters. The surface enhanced Raman spectra of a 2.75 × 10-7 M concentration of Cresyl Violet in ethanol were obtained using these photographic substrates. The Raman and resonant Raman enhancement of Cresyl Violet varies from substrate to substrate, as does the ratio of Raman to resonant Raman peak heights.

Effect of conformational mobility and hydrogen-bonding interactions on the selectivity of some guanidinoaryl-substituted mechanism-based inhibitors of trypsin-like serine proteases.

PubMed

Rai, R; Katzenellenbogen, J A

1992-11-13

Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.

ACE phenotyping in Gaucher disease.

PubMed

Danilov, Sergei M; Tikhomirova, Victoria E; Metzger, Roman; Naperova, Irina A; Bukina, Tatiana M; Goker-Alpan, Ozlem; Tayebi, Nahid; Gayfullin, Nurshat M; Schwartz, David E; Samokhodskaya, Larisa M; Kost, Olga A; Sidransky, Ellen

2018-04-01

Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen. The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Three-Dimensional Solid-State Lithium-Ion Batteries Fabricated by Conformal Vapor-Phase Chemistry.

PubMed

Pearse, Alexander; Schmitt, Thomas; Sahadeo, Emily; Stewart, David M; Kozen, Alexander; Gerasopoulos, Konstantinos; Talin, A Alec; Lee, Sang Bok; Rubloff, Gary W; Gregorczyk, Keith E

2018-05-22

Three-dimensional thin-film solid-state batteries (3D TSSB) were proposed by Long et al. in 2004 as a structure-based approach to simultaneously increase energy and power densities. Here, we report experimental realization of fully conformal 3D TSSBs, demonstrating the simultaneous power-and-energy benefits of 3D structuring. All active battery components-electrodes, solid electrolyte, and current collectors-were deposited by atomic layer deposition (ALD) onto standard CMOS processable silicon wafers microfabricated to form arrays of deep pores with aspect ratios up to approximately 10. The cells utilize an electrochemically prelithiated LiV 2 O 5 cathode, a very thin (40-100 nm) Li 2 PO 2 N solid electrolyte, and a SnN x anode. The fabrication process occurs entirely at or below 250 °C, promising compatibility with a variety of substrates as well as integrated circuits. The multilayer battery structure enabled all-ALD solid-state cells to deliver 37 μAh/cm 2 ·μm (normalized to cathode thickness) with only 0.02% per-cycle capacity loss. Conformal fabrication of full cells over 3D substrates increased the areal discharge capacity by an order of magnitude while simulteneously improving power performance, a trend consistent with a finite element model. This work shows that the exceptional conformality of ALD, combined with conventional semiconductor fabrication methods, provides an avenue for the successful realization of long-sought 3D TSSBs which provide power performance scaling in regimes inaccessible to planar form factor cells.

X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

NASA Astrophysics Data System (ADS)

Prokofev, I. I.; Lashkov, A. A.; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

2016-11-01

In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2'-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

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

Prokofev, I. I.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdulkhakov, A. G.

In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data,more » supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2′-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation.« less

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

PubMed Central

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

A Novel Angiotensin I-Converting Enzyme Mutation (S333W) Impairs N-Domain Enzymatic Cleavage of the Anti-Fibrotic Peptide, AcSDKP

PubMed Central

Danilov, Sergei M.; Wade, Michael S.; Schwager, Sylva L.; Douglas, Ross G.; Nesterovitch, Andrew B.; Popova, Isolda A.; Hogarth, Kyle D.; Bhardwaj, Nakul; Schwartz, David E.; Sturrock, Edward D.; Garcia, Joe G. N.

2014-01-01

Background Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. Results We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis. PMID:24505347

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting results of other DNA sequences (Table 2S); calculation of dissociation constants (Fig. 3S, 4S; Table 2S); geometric model for quantitation of DNA bending angle induced by specific IHF binding (Fig. 4S); customized flow cell assembly (Fig. 5S); real-time measurement of average fluorophore height change by SSFM (Fig. 6S); summary of binding parameters obtained from additive isotherm model fitting (Table 3S); average surface densities of 10 dsDNA spots and bound IHF at equilibrium (Table 4S); effects of surface densities on the binding and bending of dsDNA (Tables 5S, 6S and Fig. 7S-10S). See DOI: 10.1039/c5nr06785e

The Detrimental Effects of Oxytocin-Induced Conformity on Dishonesty in Competition.

PubMed

Aydogan, Gökhan; Jobst, Andrea; D'Ardenne, Kimberlee; Müller, Norbert; Kocher, Martin G

2017-06-01

Justifications may promote unethical behavior because they constitute a convenient loophole through which people can gain from immoral behavior and preserve a positive self-image at the same time. A justification that is widely used is rooted in conformity: Unethical choices become more permissible because one's peers are expected to make the same unethical choices. In the current study, we tested whether an exogenous alteration of conformity led to a lower inclination to adhere to a widely accepted norm (i.e., honesty) under the pressure of competition. We took advantage of the well-known effects of intranasally applied oxytocin on affiliation, in-group conformity, and in-group favoritism in humans. We found that conformity was enhanced by oxytocin, and this enhancement had a detrimental effect on honesty in a competitive environment but not in a noncompetitive environment. Our findings contribute to recent evidence showing that competition may lead to unethical behavior and erode moral values.

Enhanced light output from the nano-patterned InP semiconductor substrate through the nanoporous alumina mask.

PubMed

Jung, Mi; Kim, Jae Hun; Lee, Seok; Jang, Byung Jin; Lee, Woo Young; Oh, Yoo-Mi; Park, Sun-Woo; Woo, Deokha

2012-07-01

A significant enhancement in the light output from nano-patterned InP substrate covered with a nanoporous alumina mask was observed. A uniform nanohole array on an InP semiconductor substrate was fabricated by inductively coupled plasma reactive ion etching (ICP-RIE), using the nanoporous alumina mask as a shadow mask. The light output property of the semiconductor substrate was investigated via photoluminescence (PL) intensity measurement. The InP substrate with a nanohole array showed a more enhanced PL intensity compared with the raw InP substrate without a nanohole structure. After ICP-RIE etching, the light output from the nanoporous InP substrate covered with a nanoporous alumina mask showed fourfold enhanced PL intensity compared with the raw InP substrate. These results can be used as a prospective method for increasing the light output efficiency of optoelectronic devices.

Superhydrophobic diatomaceous earth

DOEpatents

Simpson, John T [Clinton, TN; D& #x27; Urso, Brian R [Clinton, TN

2012-07-10

A superhydrophobic powder is prepared by coating diatomaceous earth (DE) with a hydrophobic coating on the particle surface such that the coating conforms to the topography of the DE particles. The hydrophobic coating can be a self assembly monolayer of a perfluorinated silane coupling agent. The DE is preferably natural-grade DE where organic impurities have been removed. The superhydrophobic powder can be applied as a suspension in a binder solution to a substrate to produce a superhydrophobic surface on the substrate.

Thermoinactivation analysis of vacuolar H(+)-pyrophosphatase.

PubMed

Yang, Su J; Jiang, Shih S; Hsiao, Yi Y; Van, Ru C; Pan, Yih J; Pan, Rong L

2004-06-07

Vacuolar H(+)-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) catalyzes both the hydrolysis of PP(i) and the electrogenic translocation of proton from the cytosol to the lumen of the vacuole. Vacuolar H(+)-PPase, purified from etiolated hypocotyls of mung bean (Vigna radiata L.), is a homodimer with a molecular mass of 145 kDa. To investigate the relationship between structure and function of this H(+)-translocating enzyme, thermoinactivation analysis was employed. Thermoinactivation studies suggested that vacuolar H(+)-PPase consists of two distinct states upon heat treatment and exhibited different transition temperatures in the presence and absence of ligands (substrate and inhibitors). Substrate protection of H(+)-PPase stabilizes enzyme structure by increasing activation energy from 54.9 to 70.2 kJ/mol. We believe that the conformation of this enzyme was altered in the presence of substrate to protect against the thermoinactivation. In contrast, the modification of H(+)-PPase by inhibitor (fluorescein 5'-isothiocyanate; FITC) augmented the inactivation by heat treatment. The native, substrate-bound, and FITC-labeled vacuolar H(+)-PPases possess probably distinct conformation and show different modes of susceptibility to thermoinactivation. Our results also indicate that the structure of one subunit of this homodimer exerts long distance effect on the other, suggesting a specific subunit-subunit interaction in vacuolar H(+)-PPase. A working model was proposed to interpret the relationship of the structure and function of vacuolar H(+)-PPase.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

PubMed

Hamilton, P J; Campbell, N G; Sharma, S; Erreger, K; Herborg Hansen, F; Saunders, C; Belovich, A N; Sahai, M A; Cook, E H; Gether, U; McHaourab, H S; Matthies, H J G; Sutcliffe, J S; Galli, A

2013-12-01

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Hairy and Slippery Polyoxazoline-Based Copolymers on Model and Cartilage Surfaces.

PubMed

Morgese, Giulia; Ramakrishna, Shivaprakash N; Simic, Rok; Zenobi-Wong, Marcy; Benetti, Edmondo M

2018-02-12

Comb-like polymers presenting a hydroxybenzaldehyde (HBA)-functionalized poly(glutamic acid) (PGA) backbone and poly(2-methyl-2-oxazoline) (PMOXA) side chains chemisorb on aminolized substrates, including cartilage surfaces, forming layers that reduce protein contamination and provide lubrication. The structure, physicochemical, biopassive, and tribological properties of PGA-PMOXA-HBA films are finely determined by the copolymer architecture, its reactivity toward the surface, i.e. PMOXA side-chain crowding and HBA density, and by the copolymer solution concentration during assembly. Highly reactive species with low PMOXA content form inhomogeneous layers due to the limited possibility of surface rearrangements by strongly anchored copolymers, just partially protecting the functionalized surface from protein contamination and providing a relatively weak lubrication on cartilage. Biopassivity and lubrication can be improved by increasing copolymer concentration during assembly, leading to a progressive saturation of surface defects across the films. In a different way, less reactive copolymers presenting high PMOXA side-chain densities form uniform, biopassive, and lubricious films, both on model aminolized silicon oxide surfaces, as well as on cartilage substrates. When assembled at low concentrations these copolymers adopt a "lying down" conformation, i.e. adhering via their backbones onto the substrates, while at high concentrations they undergo a conformational transition, assuming a more densely packed, "standing up" structure, where they stretch perpendicularly from the substrate. This specific arrangement reduces protein contamination and improves lubrication both on model as well as on cartilage surfaces.

Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study.

PubMed

McClory, James; Timson, David J; Singh, Warispreet; Zhang, Jian; Huang, Meilan

2017-12-14

Isopentenyl phosphate kinase (IPK) catalyzes the Mg 2+ -ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg 2+ and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

Decoupling optical function and geometrical form using conformal flexible dielectric metasurfaces

NASA Astrophysics Data System (ADS)

Kamali, Seyedeh Mahsa; Arbabi, Amir; Arbabi, Ehsan; Horie, Yu; Faraon, Andrei

2016-05-01

Physical geometry and optical properties of objects are correlated: cylinders focus light to a line, spheres to a point and arbitrarily shaped objects introduce optical aberrations. Multi-functional components with decoupled geometrical form and optical function are needed when specific optical functionalities must be provided while the shapes are dictated by other considerations like ergonomics, aerodynamics or aesthetics. Here we demonstrate an approach for decoupling optical properties of objects from their physical shape using thin and flexible dielectric metasurfaces which conform to objects' surface and change their optical properties. The conformal metasurfaces are composed of silicon nano-posts embedded in a polymer substrate that locally modify near-infrared (λ=915 nm) optical wavefronts. As proof of concept, we show that cylindrical lenses covered with metasurfaces can be transformed to function as aspherical lenses focusing light to a point. The conformal metasurface concept is highly versatile for developing arbitrarily shaped multi-functional optical devices.

Neurotransmitter and psychostimulant recognition by the dopamine transporter

PubMed Central

Wang, Kevin H.; Penmatsa, Aravind; Gouaux, Eric

2015-01-01

Na+/Cl−-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine x-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine (DA), a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants D-amphetamine, methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters. PMID:25970245

Kinetics of Mixed Microbial Assemblages Enhance Removal of Highly Dilute Organic Substrates

PubMed Central

Lewis, David L.; Hodson, Robert E.; Hwang, Huey-Min

1988-01-01

Our experiments with selected organic substrates reveal that the rate-limiting process governing microbial degradation rates changes with substrate concentration, S, in such a manner that substrate removal is enhanced at lower values of S. This enhancement is the result of the dominance of very efficient systems for substrate removal at low substrate concentrations. The variability of dominant kinetic parameters over a range of S causes the kinetics of complex assemblages to be profoundly dissimilar to those of systems possessing a single set of kinetic parameters; these findings necessitate taking a new approach to predicting substrate removal rates over wide ranges of S. PMID:16347715

Large area nanoimprint by substrate conformal imprint lithography (SCIL)

NASA Astrophysics Data System (ADS)

Verschuuren, Marc A.; Megens, Mischa; Ni, Yongfeng; van Sprang, Hans; Polman, Albert

2017-06-01

Releasing the potential of advanced material properties by controlled structuring materials on sub-100-nm length scales for applications such as integrated circuits, nano-photonics, (bio-)sensors, lasers, optical security, etc. requires new technology to fabricate nano-patterns on large areas (from cm2 to 200 mm up to display sizes) in a cost-effective manner. Conventional high-end optical lithography such as stepper/scanners is highly capital intensive and not flexible towards substrate types. Nanoimprint has had the potential for over 20 years to bring a cost-effective, flexible method for large area nano-patterning. Over the last 3-4 years, nanoimprint has made great progress towards volume production. The main accelerator has been the switch from rigid- to wafer-scale soft stamps and tool improvements for step and repeat patterning. In this paper, we discuss substrate conformal imprint lithography (SCIL), which combines nanometer resolution, low patterns distortion, and overlay alignment, traditionally reserved for rigid stamps, with the flexibility and robustness of soft stamps. This was made possible by a combination of a new soft stamp material, an inorganic resist, combined with an innovative imprint method. Finally, a volume production solution will be presented, which can pattern up to 60 wafers per hour.

Performance-Enhancing Methods for Au Film over Nanosphere Surface-Enhanced Raman Scattering Substrate and Melamine Detection Application

PubMed Central

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm−1 and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect. PMID:24886913

Performance-enhancing methods for Au film over nanosphere surface-enhanced Raman scattering substrate and melamine detection application.

PubMed

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm(-1) and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect.

Evaluation of the effects of a freeze/thaw environment on cellular glass

NASA Technical Reports Server (NTRS)

Frickland, P.; Cleland, E.; Hasegawa, T.

1981-01-01

Using the evaluation criteria of water vapor permeability and conformability, a protective butylrubber/silicone conformal coating system was selected for use on Foamglas substrates in a freeze/thaw environment. The selection of a specific freeze/thaw cycle which closely models field conditions is discussed. A sampling plan is described which allows independent evaluation of the effects of conformal coatings, cycle number and location within the environmental chamber. The results of visual examination, measurement of density, modulus of rupture and Young's modulus are reported. Based upon statistical evaluation of the experimental results, it is concluded that no degradation in mechanical properties of either coated or uncoated Foamglas occurred within the duration of the test (53 freeze/thaw cycles).

Quantitative Comparison of Protein Adsorption and Conformational Changes on Dielectric-Coated Nanoplasmonic Sensing Arrays.

PubMed

Ferhan, Abdul Rahim; Jackman, Joshua A; Sut, Tun Naw; Cho, Nam-Joon

2018-04-22

Nanoplasmonic sensors are a popular, surface-sensitive measurement tool to investigate biomacromolecular interactions at solid-liquid interfaces, opening the door to a wide range of applications. In addition to high surface sensitivity, nanoplasmonic sensors have versatile surface chemistry options as plasmonic metal nanoparticles can be coated with thin dielectric layers. Within this scope, nanoplasmonic sensors have demonstrated promise for tracking protein adsorption and substrate-induced conformational changes on oxide film-coated arrays, although existing studies have been limited to single substrates. Herein, we investigated human serum albumin (HSA) adsorption onto silica- and titania-coated arrays of plasmonic gold nanodisks by localized surface plasmon resonance (LSPR) measurements and established an analytical framework to compare responses across multiple substrates with different sensitivities. While similar responses were recorded on the two substrates for HSA adsorption under physiologically-relevant ionic strength conditions, distinct substrate-specific behavior was observed at lower ionic strength conditions. With decreasing ionic strength, larger measurement responses occurred for HSA adsorption onto silica surfaces, whereas HSA adsorption onto titania surfaces occurred independently of ionic strength condition. Complementary quartz crystal microbalance-dissipation (QCM-D) measurements were also performed, and the trend in adsorption behavior was similar. Of note, the magnitudes of the ionic strength-dependent LSPR and QCM-D measurement responses varied, and are discussed with respect to the measurement principle and surface sensitivity of each technique. Taken together, our findings demonstrate how the high surface sensitivity of nanoplasmonic sensors can be applied to quantitatively characterize protein adsorption across multiple surfaces, and outline broadly-applicable measurement strategies for biointerfacial science applications.

Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism

PubMed Central

Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar; Wierdl, Monika; Gwaltney, Steven R.; Potter, Philip M.; Wadkins, Randy M.

2013-01-01

Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/β fold family of hydrolases, of which this CE is a member. PMID:22127613

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.

Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases.

PubMed

Khurana, S; Sanli, G; Powers, D B; Anderson, S; Blaber, M

2000-04-01

2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75. Copyright 2000 Wiley-Liss, Inc.

Local entropy difference upon a substrate binding of a psychrophilic α-amylase and a mesophilic homologue

NASA Astrophysics Data System (ADS)

Kosugi, Takahiro; Hayashi, Shigehiko

2011-01-01

Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.

Substrate-Independent Epitaxial Growth of the Metal-Organic Framework MOF-508a.

PubMed

Wilson, M; Barrientos-Palomo, S N; Stevens, P C; Mitchell, N L; Oswald, G; Nagaraja, C M; Badyal, J P S

2018-01-31

Plasmachemical deposition is a substrate-independent method for the conformal surface functionalization of solid substrates. Structurally well-defined pulsed plasma deposited poly(1-allylimidazole) layers provide surface imidazole linker groups for the directed liquid-phase epitaxial (layer-by-layer) growth of metal-organic frameworks (MOFs) at room temperature. For the case of microporous [Zn (benzene-1,4-dicarboxylate)-(4,4'-bipyridine) 0.5 ] (MOF-508), the MOF-508a polymorph containing two interpenetrating crystal lattice frameworks undergoes orientated Volmer-Weber growth and displays CO 2 gas capture behavior at atmospheric concentrations in proportion to the number of epitaxially grown MOF-508 layers.

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.

Effect of bisecting GlcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution.

PubMed

Nishima, Wataru; Miyashita, Naoyuki; Yamaguchi, Yoshiki; Sugita, Yuji; Re, Suyong

2012-07-26

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

Molecular Level Characterization of the Structure and Interactions in Peptide-Functionalized Metal-Organic Frameworks.

PubMed

Todorova, Tanya K; Rozanska, Xavier; Gervais, Christel; Legrand, Alexandre; Ho, Linh N; Berruyer, Pierrick; Lesage, Anne; Emsley, Lyndon; Farrusseng, David; Canivet, Jérôme; Mellot-Draznieks, Caroline

2016-11-07

We use density functional theory, newly parameterized molecular dynamics simulations, and last generation 15 N dynamic nuclear polarization surface enhanced solid-state NMR spectroscopy (DNP SENS) to understand graft-host interactions and effects imposed by the metal-organic framework (MOF) host on peptide conformations in a peptide-functionalized MOF. Focusing on two grafts typified by MIL-68-proline (-Pro) and MIL-68-glycine-proline (-Gly-Pro), we identified the most likely peptide conformations adopted in the functionalized hybrid frameworks. We found that hydrogen bond interactions between the graft and the surface hydroxyl groups of the MOF are essential in determining the peptides conformation(s). DNP SENS methodology shows unprecedented signal enhancements when applied to these peptide-functionalized MOFs. The calculated chemical shifts of selected MIL-68-NH-Pro and MIL-68-NH-Gly-Pro conformations are in a good agreement with the experimentally obtained 15 N NMR signals. The study shows that the conformations of peptides when grafted in a MOF host are unlikely to be freely distributed, and conformational selection is directed by strong host-guest interactions. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1.

PubMed

Hwa, Kuo Yuan; Subramani, Boopathi; Shen, San-Tai; Lee, Yu-May

2015-09-01

β-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with β-glucosidase, β-mannosidase, β-fucosidase and β-galactosidase activities. Sequence alignment with other β-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic β-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its β-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for β-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures. Copyright © 2015 Elsevier Inc. All rights reserved.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

PubMed

Ulfig, Agnes; Freudl, Roland

2018-05-11

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

PDMS based multielectrode arrays for superior in-vitro retinal stimulation and recording.

PubMed

Biswas, Satarupa; Sikdar, Debdeep; Das, Debanjan; Mahadevappa, Manjunatha; Das, Soumen

2017-08-25

Understanding of the neural response to electrical stimulation requires simultaneous recording from the various neurons of retina. Electrodes form the physical interface with the neural or retinal tissue. Successful retinal stimulation and recording demands conformal integration of these electrodes with the soft tissue to ensure establishment of proper electrical connection with the excitable tissue. Mechanical impedance of polydimethylsiloxane (PDMS) being compliant with that of retinal tissue, offers excellent potential as a substrate for metal electrodes. In this paper, Cr/Au micro electrodes with 200 μm diameter were fabricated on rigid and flexible PDMS substrates under crack free condition. Spontaneous buckling of thin films over PDMS substrates improved electrode performance circumventing the fabrication issues faced over a buckled surface. Individual electrodes from the multielectrode arrays (MEAs) were examined with electrochemical impedance spectroscopy and cyclic voltammetry. Controlled fabrication process as described here generates buckles in the metal films leading to increased electrode surface area that increases the charge storage capacity and decreases the interface impedance of the metal electrodes. At 1 kHz, impedance was reduced from 490 ± 27 kΩ to 246 ± 19 kΩ and charge storage capacity was increased from 0.40 ± 0.87 mC/cm 2 to 2.1 ± 0.87 mC/cm 2 . Neural spikes recorded with PDMS based electrodes from isolated retina also contained less noise as indicated by signal to noise ratio analysis. The present study established that the use of PDMS as a substrate for MEAs can enhance the performance of any thin film metal electrodes without incorporation of any coating layers or nanomaterials.

Dynamic Substrate for the Physical Encoding of Sensory Information in Bat Biosonar

NASA Astrophysics Data System (ADS)

Müller, Rolf; Gupta, Anupam K.; Zhu, Hongxiao; Pannala, Mittu; Gillani, Uzair S.; Fu, Yanqing; Caspers, Philip; Buck, John R.

2017-04-01

Horseshoe bats have dynamic biosonar systems with interfaces for ultrasonic emission (reception) that change shape while diffracting the outgoing (incoming) sound waves. An information-theoretic analysis based on numerical and physical prototypes shows that these shape changes add sensory information (mutual information between distant shape conformations <20 %), increase the number of resolvable directions of sound incidence, and improve the accuracy of direction finding. These results demonstrate that horseshoe bats have a highly effective substrate for dynamic encoding of sensory information.

Dynamic Substrate for the Physical Encoding of Sensory Information in Bat Biosonar.

PubMed

Müller, Rolf; Gupta, Anupam K; Zhu, Hongxiao; Pannala, Mittu; Gillani, Uzair S; Fu, Yanqing; Caspers, Philip; Buck, John R

2017-04-14

Horseshoe bats have dynamic biosonar systems with interfaces for ultrasonic emission (reception) that change shape while diffracting the outgoing (incoming) sound waves. An information-theoretic analysis based on numerical and physical prototypes shows that these shape changes add sensory information (mutual information between distant shape conformations <20%), increase the number of resolvable directions of sound incidence, and improve the accuracy of direction finding. These results demonstrate that horseshoe bats have a highly effective substrate for dynamic encoding of sensory information.

A Broadband High-Gain Bi-Layer Log-Periodic Dipole Array (LPDA) for Ultra High Frequency (UHF) Conformal Load Bearing Antenna Structures (CLAS) Applications

DTIC Science & Technology

2014-08-01

AFRL-RQ-WP-TR-2014-0212 University of South Carolina Department of Electrical Engineering Columbia, SC 29208 Structures Technology Branch...S2603-04-C01. Cleared for Public Release - Case Number: . Nicholas Bishop and M. Ali are with the Department of Electrical Engineering, University of...Lower substrate Upper substrate Foam core Coax Feed tube LPDA traces Coax inner conductor Feed tube Copper plate Input 88ABW-2014-3668, 8

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Toward a mechanistic understanding of patterns in biomineralization and new insights for old dogmas in geological settings (Invited)

NASA Astrophysics Data System (ADS)

Dove, P. M.; Hamm, L.; Giuffre, A. J.; Han, N.; De Yoreo, J. J.

2013-12-01

The ability of organisms to mineralize tissues into skeletons and other functional structures is a remarkable achievement of biology. Yet, the physical basis for how macromolecules regulate the placement and onset of mineral formation is not well established. Efforts to understand nucleation onto organic substrates have produced two, seemingly contradictory, lines of thought: The biomineralization community widely assumes the organic matrix promotes nucleation through stereochemical matching to guide the organization of solute ions, while materials synthesis groups use simple binding assays to correlate high binding strength with good promoters of nucleation. This study reconciles the two views and provides a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate binding free energies. Using surface assembled monolayers (SAM) as simple model systems, we first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, hydroxyl) and conformations (C11, C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate (ACC). Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcite-substrate binding free energies, ΔGb. Moreover, we show that within the classical theory of nucleation, γ and ΔGb should be linearly related. The results bear out this prediction and demonstrate that low energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings reconcile the long-standing concept of templated nucleation through stereochemical matching with the conventional wisdom that ';good binders are good nucleators'. Alternative perspectives become internally consistent when viewed through the lens of crystal-substrate binding and provide a physical basis for how organic chemistry can direct temporal and spatial patterns of carbonate nucleation.

Molecular propulsion: chemical sensing and chemotaxis of DNA driven by RNA polymerase.

PubMed

Yu, Hua; Jo, Kyubong; Kounovsky, Kristy L; de Pablo, Juan J; Schwartz, David C

2009-04-29

Living cells sense extracellular signals and direct their movements in response to stimuli in environment. Such autonomous movement allows these machines to sample chemical change over a distance, leading to chemotaxis. Synthetic catalytic rods have been reported to chemotax toward hydrogen peroxide fuel. Nevertheless individualized autonomous control of movement of a population of biomolecules under physiological conditions has not been demonstrated. Here we show the first experimental evidence that a molecular complex consisting of a DNA template and associating RNA polymerases (RNAPs) displays chemokinetic motion driven by transcription substrates nucleoside triphosphates (NTPs). Furthermore this molecular complex exhibits a biased migration into a concentration gradient of NTPs, resembling chemotaxis. We describe this behavior as "Molecular Propulsion", in which RNAP transcriptional actions deform DNA template conformation engendering measurable enhancement of motility. Our results provide new opportunities for designing and directing nanomachines by imposing external triggers within an experimental system.

Identification of key residues for protein conformational transition using elastic network model.

PubMed

Su, Ji Guo; Xu, Xian Jin; Li, Chun Hua; Chen, Wei Zu; Wang, Cun Xin

2011-11-07

Proteins usually undergo conformational transitions between structurally disparate states to fulfill their functions. The large-scale allosteric conformational transitions are believed to involve some key residues that mediate the conformational movements between different regions of the protein. In the present work, a thermodynamic method based on the elastic network model is proposed to predict the key residues involved in protein conformational transitions. In our method, the key functional sites are identified as the residues whose perturbations largely influence the free energy difference between the protein states before and after transition. Two proteins, nucleotide binding domain of the heat shock protein 70 and human/rat DNA polymerase β, are used as case studies to identify the critical residues responsible for their open-closed conformational transitions. The results show that the functionally important residues mainly locate at the following regions for these two proteins: (1) the bridging point at the interface between the subdomains that control the opening and closure of the binding cleft; (2) the hinge region between different subdomains, which mediates the cooperative motions between the corresponding subdomains; and (3) the substrate binding sites. The similarity in the positions of the key residues for these two proteins may indicate a common mechanism in their conformational transitions.

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

Light Coupling and Trapping in Ultrathin Cu(In,Ga)Se2 Solar Cells Using Dielectric Scattering Patterns.

PubMed

van Lare, Claire; Yin, Guanchao; Polman, Albert; Schmid, Martina

2015-10-27

We experimentally demonstrate photocurrent enhancement in ultrathin Cu(In,Ga)Se2 (CIGSe) solar cells with absorber layers of 460 nm by nanoscale dielectric light scattering patterns printed by substrate conformal imprint lithography. We show that patterning the front side of the device with TiO2 nanoparticle arrays results in a small photocurrent enhancement in almost the entire 400-1200 nm spectral range due to enhanced light coupling into the cell. Three-dimensional finite-difference time-domain simulations are in good agreement with external quantum efficiency measurements. Patterning the Mo/CIGSe back interface using SiO2 nanoparticles leads to strongly enhanced light trapping, increasing the efficiency from 11.1% for a flat to 12.3% for a patterned cell. Simulations show that optimizing the array geometry could further improve light trapping. Including nanoparticles at the Mo/CIGSe interface leads to substantially reduced parasitic absorption in the Mo back contact. Parasitic absorption in the back contact can be further reduced by fabricating CIGSe cells on top of a SiO2-patterned In2O3:Sn (ITO) back contact. Simulations show that these semitransparent cells have similar spectrally averaged reflection and absorption in the CIGSe active layer as a Mo-based patterned cell, demonstrating that the absorption losses in the Mo can be partially turned into transmission through the semitransparent geometry.

Single molecule imaging of conformational dynamics in sodium-coupled transporters

NASA Astrophysics Data System (ADS)

Terry, Daniel S.

Neurotransmitter:sodium symporter (NSS) proteins remove neurotransmitters released into the synapse through a transport process driven by the physiological sodium ion (Na+) gradient. NSSs for dopamine, noradrenaline, and serotonin are targeted by the psychostimulants cocaine and amphetamines, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed the NSS molecular architecture and has been the basis for extensive structural, biochemical, and computational investigations of the mechanism of transporter proteins with a LeuT-like fold. In this dissertation, the conformational states sampled by LeuT are explored using single-molecule fluorescence resonance energy transfer imaging methods, with special focus on the motions of transmembrane helix 1a that lead to inward release of substrate. We also explored how dynamics are modulated by substrate, Na+, and protons to produce efficient transport. These advances represent a first of a kind study of the dynamics of an integral membrane protein at a truly single-molecule scale. Advances in instrumentation, analysis tools, and organic fluorophores were all required to achieve these goals, and such advances are also described. While these experiments were performed with detergent-solubilized protein, preliminary work suggests that imaging of LeuT in proteoliposomes is feasible and a fluorescence sensor assay could be used to simultaneously detect conformational dynamics and transport function.

The role of nonbonded interactions in the conformational dynamics of organophosphorous hydrolase adsorbed onto functionalized mesoporous silica surfaces.

PubMed

Gomes, Diego E B; Lins, Roberto D; Pascutti, Pedro G; Lei, Chenghong; Soares, Thereza A

2010-01-14

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase*

PubMed Central

Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Álvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

2010-01-01

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism. PMID:20056599

Structure of GroEL in complex with an early folding intermediate of alanine glyoxylate aminotransferase.

PubMed

Albert, Armando; Yunta, Cristina; Arranz, Rocío; Peña, Alvaro; Salido, Eduardo; Valpuesta, José María; Martín-Benito, Jaime

2010-02-26

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.

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

Importance of cholesterol in dopamine transporter function

PubMed Central

Jones, Kymry T.; Zhen, Juan; Reith, Maarten E.A.

2012-01-01

The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared to non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl-β-cyclodextrin (mβCD), which effectively depletes total membrane cholesterol- uniformly affecting cholesterol-DAT interactions in both raft and non-raft membrane domains- reduced both DA uptake and efflux rate. In contrast, disruption of raft localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft-localized DAT in substrate uptake or efflux. Supra-normal repletion of cholesterol depleted cells with the analogue desmosterol, a non-raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn2+ and the conformationally-biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward-facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol-DAT interactions in regulating DAT function. PMID:22957537

Integration of amorphous tantalum silicon nitride (TaSiN) films as diffusion barriers in a Cu/SiLK(TM) metallization scheme

NASA Astrophysics Data System (ADS)

Padiyar, Sumant Devdas

2003-09-01

Current and future performance requirements for high- speed integrated circuit (IC) devices have placed great emphasis on the introduction of novel materials, deposition techniques and improved metrology techniques. The introduction of copper interconnects and more currently low-k dielectric materials in IC fabrication are two such examples. This introduction necessitates research on the compatibility of these materials and process techniques with adjacent diffusion barrier materials. One candidate, which has attracted significant attention is tantalum-silicon-nitride (TaSiN) on account of its superior diffusion barrier performance and high recrystallization temperature1. The subject of this dissertation is an investigation of the integration compatibility and performance of TaSiN barrier layers with a low-k dielectric polymer (SiLK ®2). A plasma- enhanced chemical vapor deposition (PECVD) approach is taken for growth of TaSiN films in this work due to potential advantages in conformal film coverage compared to more conventional physical vapor deposition methods. A Design of Experiment (DOE) methodology was introduced for PECVD of TaSiN on SiLK to optimize film properties such as film composition, resistivity, growth rate and film roughness with respect to the predictors viz. substrate temperature, precursor gas flow and plasma power. The first pass study determined the response window for optimized TaSiN film composition, growth rate and low halide contamination and the compatibility of the process with an organic polymer substrate, i.e. SiLK. Second-pass studies were carried out to deposit ultra- thin (10nm) films on: (a)blanket SiLK to investigate the performance of TaSiN films against copper diffusion, and (b)patterned SiLK to evaluate step coverage and conformality. All TaSiN depositions were carried out on SiO2 substrates for baseline comparisons. A second purpose of the diffusion barrier in IC processing is to improve interfacial adhesion between the barrier and the adjacent dielectric material; especially important for an organic polymer like SiLK. Hence, a detailed study was undertaken to evaluate the interfacial adhesion of TaSiN with SiLK and SiO2 and study the dependence of the adhesion with the film composition. The results of diffusion barrier performance studies, conformality studies, and interfacial adhesion studies of TaSiN films are discussed in relation to the elemental compositions of the films. 1J. S. Reid, M. Nicolet, J. Appl. Phys. 79 (2) p. 1109 (1996). 2SiLK is a low-k dielectric candidate registered by Dow Chemical Company, MI.

NMR Mapping of Protein Conformational Landscapes using Coordinated Behavior of Chemical Shifts upon Ligand Binding

PubMed Central

Cembran, Alessandro; Kim, Jonggul; Gao, Jiali; Veglia, Gianluigi

2014-01-01

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria following linear trajectories of NMR chemical shifts. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues’ response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein’s approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins. PMID:24604024

Critical assessment of enhancement factor measurements in surface-enhanced Raman scattering on different substrates.

PubMed

Rodrigues, Daniel C; de Souza, Michele L; Souza, Klester S; dos Santos, Diego P; Andrade, Gustavo F S; Temperini, Marcia L A

2015-09-07

The SERS enhancement factor (SERS-EF) is one of the most important parameters that characterizes the ability of a given substrate to enhance the Raman signal for SERS applications. The comparison of SERS intensities and SERS-EF values across different substrates is a common practice to unravel the performance of a given substrate. In this study, it is shown that such a comparison may lack significance if we compare substrates of very distinct nature and optical properties. It is specifically shown that the SERS-EF values for static substrates (e.g. immobilized metallic nanostructures) cannot be compared to those of dynamic ones (e.g. colloidal metal nanoparticle solutions), and that the optical properties for the latter show strong dependence on the metal-molecule interaction dynamics. The most representative experimental results concerning the dynamic substrates have been supported by generalized Mie theory simulations, which are tools used to describe the substrate complexity and the microscopic information not usually taken into account.

Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU.

PubMed

Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G; Vickery, Larry E

2004-12-24

The ATPase activity of HscA, a specialized hsp70 molecular chaperone from Escherichia coli, is regulated by the iron-sulfur cluster assembly protein IscU and the J-type co-chaperone HscB. IscU behaves as a substrate for HscA, and HscB enhances the binding of IscU to HscA. To better understand the mechanism by which HscB and IscU regulate HscA, we examined binding of HscB to the different conformational states of HscA and the effects of HscB and IscU on the kinetics of the individual steps of the HscA ATPase reaction cycle. Affinity sensor studies revealed that whereas IscU binds both ADP (R-state) and ATP (T-state) HscA complexes, HscB interacts only with an ATP-bound state. Studies of ATPase activity under single-turnover and rapid mixing conditions showed that both IscU and HscB interact with the low peptide affinity T-state of HscA (HscA++.ATP) and that both modestly accelerate (3-10-fold) the rate-determining steps in the HscA reaction cycle, k(hyd) and k(T-->R). When present together, IscU and HscB synergistically stimulate both k(hyd) (approximately = 500-fold) and k(T-->R) (approximately = 60-fold), leading to enhanced formation of the HscA.ADP-IscU complex (substrate capture). Following ADP/ATP exchange, IscU also stimulates k(R-->T) (approximately = 50-fold) and thereby accelerates the rate at which the low peptide affinity HscA++.ATP T-state is regenerated. Because HscA nucleotide exchange is fast, the overall rate of the chaperone cycle in vivo will be determined by the availability of the IscU-HscB substrate-co-chaperone complex.

A new aptameric biosensor for cocaine based on surface-enhanced Raman scattering spectroscopy.

PubMed

Chen, Jiwei; Jiang, Jianhui; Gao, Xing; Liu, Guokun; Shen, Guoli; Yu, Ruqin

2008-01-01

The present study reports the proof of principle of a reagentless aptameric sensor based on surface-enhanced Raman scattering (SERS) spectroscopy with "signal-on" architecture using a model target of cocaine. This new aptameric sensor is based on the conformational change of the surface-tethered aptamer on a binding target that draws a certain Raman reporter in close proximity to the SERS substrate, thereby increasing the Raman scattering signal due to the local enhancement effect of SERS. To improve the response performance, the sensor is fabricated from a cocaine-templated mixed self-assembly of a 3'-terminal tetramethylrhodamine (TMR)-labeled DNA aptamer on a silver colloid film by means of an alkanethiol moiety at the 5' end. This immobilization strategy optimizes the orientation of the aptamer on the surface and facilitates the folding on the binding target. Under optimized assay conditions, one can determine cocaine at a concentration of 1 muM, which compares favorably with analogous aptameric sensors based on electrochemical and fluorescence techniques. The sensor can be readily regenerated by being washed with a buffer. These results suggest that the SERS-based transducer might create a new dimension for future development of aptameric sensors for sensitive determination in biochemical and biomedical studies.

The effect of substrate on electric field enhancement of Tip-enhanced Raman spectroscopy (TERS)

NASA Astrophysics Data System (ADS)

Bahreini, Maryam

2018-01-01

The characterization of materials down to a few-molecule level is a key challenge in nanotechnology. Raman spectroscopy is a powerful method that provides chemical information via nondestructive vibrational fingerprinting. Unfortunately, this method suffers from signal weakness which prevents the study of small quantities. Tip-enhanced Raman spectroscopy (TERS) which combines the chemical sensitivity of Raman spectroscopy (RS) with high spatial resolution of scanning probe microscopy (SPM), provides chemical images of surfaces at the nanometer scale. In this method, irradiation of an SPM tip by a focused laser beam results in enhancement of local electric field via two reasons of localized surface plasmon resonance and lightning rod effect. This enhancement leads to the enhancement in Raman intensity from the sample surface in the vicinity of tip. In all TERS measurements, samples should be located on a substrate. In this paper, the dependence of the electric field enhancement to the substrate has been investigated. In simulations, three-dimensional finite-difference time-domain (3D-FDTD) method is used for numerical solution of Maxwell's equations. Our results show that the electric field enhancement is weak for the tip alone case. Introducing a substrate provides further electric field enhancement via near field electromagnetic dipole-dipole coupling between the tip and substrate. Since the side-illumination geometry is used for laser irradiation, the vertical component of the incident field plays a dominant role in the electric field enhancement. Therefore, the coupling effect between the tip and the substrate is the key contribution to the enhancement. For the case of silicon tip and the gold substrate, the electric field enhancement is improved considerably. There is an optimal tip size for TERS because of the competing effects of the radiation damping and the surface scattering of the tip. The results show the substrate as an effective tool for the improvement of the TERS detection sensitivity.

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.

Visible-blind ultraviolet photodetectors on porous silicon carbide substrates

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

Naderi, N.; Hashim, M.R., E-mail: roslan@usm.my

2013-06-01

Highlights: • Highly reliable UV detectors are fabricated on porous silicon carbide substrates. • The optical properties of samples are enhanced by increasing the current density. • The optimized sample exhibits enhanced sensitivity to the incident UV radiation. - Abstract: Highly reliable visible-blind ultraviolet (UV) photodetectors were successfully fabricated on porous silicon carbide (PSC) substrates. High responsivity and high photoconductive gain were observed in a metal–semiconductor–metal ultraviolet photodetector that was fabricated on an optimized PSC substrate. The PSC samples were prepared via the UV-assisted photo-electrochemical etching of an n-type hexagonal silicon carbide (6H-SiC) substrate using different etching current densities. Themore » optical results showed that the current density is an outstanding etching parameter that controls the porosity and uniformity of PSC substrates. A highly porous substrate was synthesized using a suitable etching current density to enhance its light absorption, thereby improving the sensitivity of UV detector with this substrate. The electrical characteristics of fabricated devices on optimized PSC substrates exhibited enhanced sensitivity and responsivity to the incident radiation.« less

The activity of Rubisco's molecular chaperone, Rubisco activase, in leaf extracts

USDA-ARS?s Scientific Manuscript database

Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the “molecular chiropractic” activity of Rubisco activase (activase). To make the study of activas...

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational Behaviour of Azasugars Based on Mannuronic Acid.

PubMed

van Rijssel, Erwin R; Janssen, Antonius P A; Males, Alexandra; Davies, Gideon J; van der Marel, Gijsbert A; Overkleeft, Herman S; Codée, Jeroen D C

2017-07-04

A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C 4 chair conformation as opposed to the "normal" 4 C 1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H 4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ⋅CkGH47 complex, showing two conformations- 3 S 1 and 1 C 4 -for the DMJ inhibitor. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

All-dielectric ultrathin conformal metasurfaces: lensing and cloaking applications at 532 nm wavelength

NASA Astrophysics Data System (ADS)

Cheng, Jierong; Jafar-Zanjani, Samad; Mosallaei, Hossein

2016-12-01

Metasurfaces are ideal candidates for conformal wave manipulation on curved objects due to their low profiles and rich functionalities. Here we design and analyze conformal metasurfaces for practical optical applications at 532 nm visible band for the first time. The inclusions are silicon disk nanoantennas embedded in a flexible supporting layer of polydimethylsiloxane (PDMS). They behave as local phase controllers in subwavelength dimensions for successful modification of electromagnetic responses point by point, with merits of high efficiency, at visible regime, ultrathin films, good tolerance to the incidence angle and the grid stretching due to the curvy substrate. An efficient modeling technique based on field equivalence principle is systematically proposed for characterizing metasurfaces with huge arrays of nanoantennas oriented in a conformal manner. Utilizing the robust nanoantenna inclusions and benefiting from the powerful analyzing tool, we successfully demonstrate the superior performances of the conformal metasurfaces in two specific areas, with one for lensing and compensation of spherical aberration, and the other carpet cloak, both at 532 nm visible spectrum.

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

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

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

DOE PAGES

Rashid, Fahad; Harris, Paul D.; Zaher, Manal S.; ...

2017-02-23

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less

Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

PubMed Central

Rashid, Fahad; Harris, Paul D; Zaher, Manal S; Sobhy, Mohamed A; Joudeh, Luay I; Yan, Chunli; Piwonski, Hubert; Tsutakawa, Susan E; Ivanov, Ivaylo; Tainer, John A; Habuchi, Satoshi; Hamdan, Samir M

2017-01-01

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.001 PMID:28230529

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

PubMed

Bencze, László Csaba; Filip, Alina; Bánóczi, Gergely; Toşa, Monica Ioana; Irimie, Florin Dan; Gellért, Ákos; Poppe, László; Paizs, Csaba

2017-05-03

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k cat /K M value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

Substrate-insensitive atomic layer deposition of plasmonic titanium nitride films

DOE PAGES

Yu, Ing-Song; Cheng, Hsyi-En; Chang, Chun-Chieh; ...

2017-02-06

The plasmonic properties of titanium nitride (TiN) films depend on the type of substrate when using typical deposition methods such as sputtering. We show atomic layer deposition (ALD) of TiN films with very weak dependence of plasmonic properties on the substrate, which also suggests the prediction and evaluation of plasmonic performance of TiN nanostructures on arbitrary substrates under a given deposition condition. Our results also observe that substrates with more nitrogen-terminated (N-terminated) surfaces will have significant impact on the deposition rate as well as the film plasmonic properties. Furthermore, we illustrate that the plasmonic properties of ALD TiN films canmore » be tailored by simply adjusting the deposition and/or post-deposition annealing temperatures. These characteristics and the capability of conformal coating make ALD TiN films on templates ideal for applications that require the fabrication of complex 3D plasmonic nanostructures.« less

Cardiomyocytes from late embryos and neonates do optimal work and striate best on substrates with tissue-level elasticity: metrics and mathematics.

PubMed

Majkut, Stephanie F; Discher, Dennis E

2012-11-01

In this review, we discuss recent studies on the mechanosensitive morphology and function of cardiomyocytes derived from embryos and neonates. For early cardiomyocytes cultured on substrates of various stiffnesses, contractile function as measured by force production, work output and calcium handling is optimized when the culture substrate stiffness mimics that of the tissue from which the cells were obtained. This optimal contractile function corresponds to changes in sarcomeric protein conformation and organization that promote contractile ability. In light of current models for myofibillogenesis, a recent mathematical model of striation and alignment on elastic substrates helps to illuminate how substrate stiffness modulates early myofibril formation and organization. During embryonic heart formation and maturation, cardiac tissue mechanics change dynamically. Experiments and models highlighted here have important implications for understanding cardiomyocyte differentiation and function in development and perhaps in regeneration processes.

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

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.

Island growth as a growth mode in atomic layer deposition: A phenomenological model

NASA Astrophysics Data System (ADS)

Puurunen, Riikka L.; Vandervorst, Wilfried

2004-12-01

Atomic layer deposition (ALD) has recently gained world-wide attention because of its suitability for the fabrication of conformal material layers with thickness in the nanometer range. Although the principles of ALD were realized about 40 years ago, the description of many physicochemical processes that occur during ALD growth is still under development. A constant amount of material deposited in an ALD reaction cycle, that is, growth-per-cycle (GPC), has been a paradigm in ALD through decades. The GPC may vary, however, especially in the beginning of the ALD growth. In this work, a division of ALD processes to four classes is proposed, on the basis of how the GPC varies with the number of ALD reaction cycles: linear growth, substrate-enhanced growth, and substrate-inhibited growth of type 1 and type 2. Island growth is identified as a likely origin for type 2 substrate-inhibited growth, where the GPC increases and goes through a maximum before it settles to a constant value characteristic of a steady growth. A simple phenomenological model is developed to describe island growth in ALD. The model assumes that the substrate is unreactive with the ALD reactants, except for reactive defects. ALD growth is assumed to proceed symmetrically from the defects, resulting islands of a conical shape. Random deposition is the growth mode on the islands. The model allows the simulation of GPC curves, surface fraction curves, and surface roughness, with physically significant parameters. When the model is applied to the zirconium tetrachloride/water and the trimethylaluminum/water ALD processes on hydrogen-terminated silicon, the calculated GPC curves and surface fractions agree with the experiments. The island growth model can be used to assess the occurrence of island growth, the size of islands formed, and point of formation of a continuous ALD-grown film. The benefits and limitations of the model and the general characteristics of type 2 substrate-inhibited ALD are discussed.

Surface modification for interaction study with bacteria and preosteoblast cells

NASA Astrophysics Data System (ADS)

Song, Qing

Surface modification plays a pivotal role in bioengineering. Polymer coatings can provide biocompatibility and biofunctionalities to biomaterials through surface modification. In this dissertation, initiated chemical vapor deposition (iCVD) was utilized to coat two-dimensional (2D) and three-dimensional (3D) substrates with differently charged polyelectrolytes in order to generate antimicrobial and osteocompatible biomaterials. ICVD is a modified CVD technique that enables surface modification in an all-dry condition without substrate damage and solvent contamination. The free-radical polymerization allows the vinyl polymers to conformally coat on various micro- and nano-structured substrates and maintains the delicate structure of the functional groups. The vapor deposition of polycations provided antimicrobial activity to planar and porous substrates through destroying the negatively charged bacterial membrane and brought about high contact-killing efficiency (99.99%) against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Additionally, the polyampholytes synthesized by iCVD exhibited excellent antifouling performance against the adhesion of Gram-positive Listeria innocua and Gram-negative E. coli in phosphate buffered saline (PBS). Their antifouling activities were attributed to the electrostatic interaction and hydration layers that served as physical and energetic barriers to prevent bacterial adhesion. The contact-killing and antifouling polymers synthesized by iCVD can be applied to surface modification of food processing equipment and medical devices with the aim of reducing foodborne diseases and medical infections. Moreover, the charged polyelectrolyte modified 2D polystyrene surfaces displayed good osteocompatibility and enhanced osteogenesis of preosteoblast cells than the un-modified polystyrene surface. In order to promote osteoinduction of hydroxyapatite (HA) scaffolds, bioinspired polymer-controlled mineralization was conducted on the polyelectrolyte modified HA scaffolds. The mineralized scaffolds stimulated osteogenesis of preosteoblast cells compared with the control HA scaffolds. Therefore, the surface modification through vapor deposition of polyelectrolytes and polymer-controlled mineralization can improve osteoinduction of bone materials. In summary, the iCVD-mediated surface modification is a simple and promising approach to biofunctionalizing various structured substrates and generating antimicrobial and biocompatible biomaterials.

Conformational changes induced in the protein tyrosine kinase p72syk by tyrosine phosphorylation or by binding of phosphorylated immunoreceptor tyrosine-based activation motif peptides.

PubMed Central

Kimura, T; Sakamoto, H; Appella, E; Siraganian, R P

1996-01-01

A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein tyrosine kinases with multisubunit receptors that contain an approximately 18-amino-acid domain called the immunoreceptor tyrosine-based activation motif (ITAM). Tyrosine-phosphorylated Syk from activated cells was in a conformation different from that in nonstimulated cells as demonstrated by changes in immunoreactivity. The addition of tyrosine-diphosphorylated ITAM peptides resulted in a similar conformational change in Syk from nonactivated cells. The peptides based on FcepsilonRIgamma were more active than those based on Fcepsilon RIbeta. In vitro autophosphorylation of Syk was dramatically enhanced by the addition of the diphosphorylated ITAM peptides. The conformational change and the enhanced autophosphorylation required the presence of both phosphorylated tyrosines on the same molecule. These conformational changes in Syk by tyrosine phosphorylation or binding to diphosphorylated ITAM could be critical for Syk activation and downstream propagation of intracellular signals. PMID:8657120

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.

Is the standard model saved asymptotically by conformal symmetry?

NASA Astrophysics Data System (ADS)

Gorsky, A.; Mironov, A.; Morozov, A.; Tomaras, T. N.

2015-03-01

It is pointed out that the top-quark and Higgs masses and the Higgs VEV with great accuracy satisfy the relations 4 m {/H 2} = 2 m {/T 2} = v 2, which are very special and reminiscent of analogous ones at Argyres-Douglas points with enhanced conformal symmetry. Furthermore, the RG evolution of the corresponding Higgs self-interaction and Yukawa couplings λ(0) = 1/8 and y(0) = 1 leads to the free-field stable point in the pure scalar sector at the Planck scale, also suggesting enhanced conformal symmetry. Thus, it is conceivable that the Standard Model is the low-energy limit of a distinct special theory with (super?) conformal symmetry at the Planck scale. In the context of such a "scenario," one may further speculate that the Higgs particle is the Goldstone boson of (partly) spontaneously broken conformal symmetry. This would simultaneously resolve the hierarchy and Landau pole problems in the scalar sector and would provide a nearly flat potential with two almost degenerate minima at the electroweak and Planck scales.