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2011

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Iowa State University

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Articles 1 - 30 of 34

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya Dec 2011

Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya

Mechanical Engineering Publications

A cocaine-specific aptamer was used as a receptor molecule in a microcantilever-based surface stress sensor for detection of cocaine molecules. An interferometric technique that relies on measuring differential displacement between two microcantilevers (a sensing/reference pair) was utilized to measure the cocaine/aptamer binding induced surface stress changes. Sensing experiments were performed for different concentrations of cocaine from 25 to 500 μM in order to determine the sensor response as a function of cocaine concentration. In the lower concentration range from 25 to 100 μM, surface stress values increased proportionally to coverage of aptamer/cocaine complexes from 11 to 26 ...


Domain Loss Has Independently Occurred Multiple Times In Plant Terpene Synthase Evolution, Matthew L. Hillwig, Meimei Xu, Tomonobu Toyomasu, Mollie S. Tiernan, Gao Wei, Guanghong Cui, Luqi Huang, Reuben J. Peters Dec 2011

Domain Loss Has Independently Occurred Multiple Times In Plant Terpene Synthase Evolution, Matthew L. Hillwig, Meimei Xu, Tomonobu Toyomasu, Mollie S. Tiernan, Gao Wei, Guanghong Cui, Luqi Huang, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a ~220 amino acid “internal sequence element” (now recognized as the γ domain) gave rise to the precursor of modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function, demonstrating that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume ...


Parsing A Multifunctional Biosynthetic Gene Cluster From Rice: Biochemical Characterization Of Cyp71z6 & 7, Yinsheng Wu, Matthew L. Hillwig, Qiang Wang, Reuben J. Peters Nov 2011

Parsing A Multifunctional Biosynthetic Gene Cluster From Rice: Biochemical Characterization Of Cyp71z6 & 7, Yinsheng Wu, Matthew L. Hillwig, Qiang Wang, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2- hydroxylase involved in a latter step of phytocassane biosynthesis. Thus ...


A Novel Labda-7,13e-Dien-15-Ol-Producing Bifunctional Diterpene Synthase From Selaginella Moellendorffii, Sibongile Mafu, Matthew L. Hillwig, Reuben J. Peters Sep 2011

A Novel Labda-7,13e-Dien-15-Ol-Producing Bifunctional Diterpene Synthase From Selaginella Moellendorffii, Sibongile Mafu, Matthew L. Hillwig, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Vascular plants invariably contain a class II diterpene cyclase (EC 5.5.1.x), as an ent-copalyl diphosphate synthase is required for gibberellin phytohormone biosynthesis. This has provided the basis for evolution of a functionally diverse enzymatic family.[1] These biocatalysts fold their substrate, the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP), to bring the terminal three carbon-carbon double bonds into proximity with each other, and then carry out bicyclization via a protonation-initiated carbocation cascade reaction. The resulting labda-15-en-8-yl+ diphosphate intermediate is most commonly quenched by deprotonation at an exocyclic methyl, as in the production of labdadienyl/copalyl ...


Free Energies For Coarse-Grained Proteins By Integrating Multibody Statistical Contact Potentials With Entropies From Elastic Network Models, Michael T. Zimmermann, Sumudu P. Leelananda, Pawel Gniewek, Yaping Feng, Robert L. Jernigan, Andrzej Kloczkowski Jul 2011

Free Energies For Coarse-Grained Proteins By Integrating Multibody Statistical Contact Potentials With Entropies From Elastic Network Models, Michael T. Zimmermann, Sumudu P. Leelananda, Pawel Gniewek, Yaping Feng, Robert L. Jernigan, Andrzej Kloczkowski

Biochemistry, Biophysics and Molecular Biology Publications

We propose a novel method of calculation of free energy for coarse grained models of proteins by combining our newly developed multibody potentials with entropies computed from elastic network models of proteins. Multi-body potentials have been of much interest recently because they take into account three dimensional interactions related to residue packing and capture the cooperativity of these interactions in protein structures. Combining four-body non-sequential, four-body sequential and pairwise short range potentials with optimized weights for each term, our coarse-grained potential improved recognition of native structure among misfolded decoys, outperforming all other contact potentials for CASP8 decoy sets and performance ...


An Interaction Between The Walker A And D-Loop Motifs Is Critical To Atp Hydrolysis And Cooperativity In Bacteriophage T4 Rad50, Metzere Bierlein De La Rosa, Scott W. Nelson Jul 2011

An Interaction Between The Walker A And D-Loop Motifs Is Critical To Atp Hydrolysis And Cooperativity In Bacteriophage T4 Rad50, Metzere Bierlein De La Rosa, Scott W. Nelson

Biochemistry, Biophysics and Molecular Biology Publications

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the ...


Multibody Coarse-Grained Potentials For Native Structure Recognition And Quality Assessment Of Protein Models, Pawel Gniewek, Sumudu P. Leelananda, Andrzej Kolinski, Robert L. Jernigan, Andrzej Kloczkowski Jun 2011

Multibody Coarse-Grained Potentials For Native Structure Recognition And Quality Assessment Of Protein Models, Pawel Gniewek, Sumudu P. Leelananda, Andrzej Kolinski, Robert L. Jernigan, Andrzej Kloczkowski

Biochemistry, Biophysics and Molecular Biology Publications

Multi-body potentials have been of much interest recently because they take into account three dimensional interactions related to residue packing and capture the cooperativity of these interactions in protein structures. Our goal was to combine long range multi-body potentials and short range potentials to improve recognition of native structure among misfolded decoys. We optimized the weights for four-body non-sequential, four-body sequential and short range potentials in order to obtain optimal model ranking results for threading and have compared these data against results obtained with other potentials. (Twenty six different coarse-grained potentials from the Potentials ‘R’Us web server have been ...


Functional Evaluation Of Bacteriophage T4 Rad50 Signature Motif Residues, Timothy J. Herdendorf, Scott W. Nelson Jun 2011

Functional Evaluation Of Bacteriophage T4 Rad50 Signature Motif Residues, Timothy J. Herdendorf, Scott W. Nelson

Biochemistry, Biophysics and Molecular Biology Publications

The repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome, and organisms have evolved a conserved mechanism to facilitate their repair. In eukaryotes, archaea, and some bacteriophage, a complex made up of Mre11 and Rad50 (MR complex), which are a nuclease and ATPase, respectively, is involved in the initial processing of DSBs. Rad50 is a member of the ATP Binding Cassette (ABC) protein superfamily, the members of which contain an important Signature motif that acts in trans to complete the dimeric ATP binding site. To explore the functional relevance of this motif, four of ...


Transplantation Of Bdnf-Secreting Mesenchymal Stem Cells Provides Neuroprotection In Chronically Hypertensive Rat Eyes, Matthew M. Harper, Sinisa D. Grozdanic, Bas Blits, Markus H. Kuehn, Daniel Zamzow, Janice E. Buss, Randy H. Kardon, Donald S. Sakaguchi Jun 2011

Transplantation Of Bdnf-Secreting Mesenchymal Stem Cells Provides Neuroprotection In Chronically Hypertensive Rat Eyes, Matthew M. Harper, Sinisa D. Grozdanic, Bas Blits, Markus H. Kuehn, Daniel Zamzow, Janice E. Buss, Randy H. Kardon, Donald S. Sakaguchi

Genetics, Development and Cell Biology Publications

Purpose.: To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH).

Methods.: COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells ...


Deficiency Of Maize Starch-Branching Enzyme I Results In Altered Starch Fine Structure, Decreased Digestibility And Reduced Coleoptile Growth During Germination, Huan Xia, Marna Yandeau-Nelson, Donald B. Thompson, Mark J. Guiltinan May 2011

Deficiency Of Maize Starch-Branching Enzyme I Results In Altered Starch Fine Structure, Decreased Digestibility And Reduced Coleoptile Growth During Germination, Huan Xia, Marna Yandeau-Nelson, Donald B. Thompson, Mark J. Guiltinan

Biochemistry, Biophysics and Molecular Biology Publications

Background: Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line.

Results: Endosperm starch granules from the sbe1a mutant were more resistant to digestion by pancreatic aamylase, and the sbe1a mutant starch had ...


Evident And Latent Plasticity Across The Rice Diterpene Synthase Family With Potential Implications For The Evolution Of Diterpenoid Metabolism In The Cereals, Dana Morrone, Matthew L. Hillwig, Matthew E. Mead, Luke Lowry, D. Bruce Fulton, Reuben J. Peters May 2011

Evident And Latent Plasticity Across The Rice Diterpene Synthase Family With Potential Implications For The Evolution Of Diterpenoid Metabolism In The Cereals, Dana Morrone, Matthew L. Hillwig, Matthew E. Mead, Luke Lowry, D. Bruce Fulton, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

The evolution of natural products biosynthetic pathways can be envisioned to occur via a number of mechanisms. Here we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-copalyl diphosphate (CPP), which are substrates for downstream diterpene synthases. Here we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn-, or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported entsandaracopimaradiene synthase also converts syn-CPP to syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP ...


Stem-Boring Caterpillars Of Switchgrass In The Midwestern United States, Jarrad R. Prasifka, Jennifer E. Buhay, Thomas W. Sappington, Emily A. Heaton, Jeffrey D. Bradshaw, Michael E. Gray May 2011

Stem-Boring Caterpillars Of Switchgrass In The Midwestern United States, Jarrad R. Prasifka, Jennifer E. Buhay, Thomas W. Sappington, Emily A. Heaton, Jeffrey D. Bradshaw, Michael E. Gray

Entomology Publications

Lepidopteran stem borers were collected from switchgrass, Panicum virgatum L., tillers showing symptoms of infestation at seven locations in Illinois and Iowa, with additional observations made on larval and adult activity. Blastobasis repartella (Dietz) (Coleophoridae), whose only known host is switchgrass, was common in plots grown for >5 yr, whereas the polyphagous stalk borer, Papaipema nebris (Guenée) (Noctuidae), was abundant in newly established (i.e., first- and second-year) switchgrass. Haimbachia albescens Capps (Crambidae) was collected from two locations in Illinois, making switchgrass the first known host for this species. Entry holes made by B. repartella and H. albescens were usually ...


Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, Francis M. Mann, Brian C. Vanderven, Reuben J. Peters Mar 2011

Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, Francis M. Mann, Brian C. Vanderven, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ~1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by ...


Rv0989c Encodes A Novel (E)-Geranyl Diphosphate Synthase Facilitating Decaprenyl Diphosphate Biosynthesis In Mycobacterium Tuberculosis, Francis M. Mann, Jill A. Thomas, Reuben J. Peters Feb 2011

Rv0989c Encodes A Novel (E)-Geranyl Diphosphate Synthase Facilitating Decaprenyl Diphosphate Biosynthesis In Mycobacterium Tuberculosis, Francis M. Mann, Jill A. Thomas, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from ...


Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, Matthew L. Hillwig, Francis M. Mann, Reuben J. Peters Feb 2011

Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, Matthew L. Hillwig, Francis M. Mann, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Most types of ambers are naturally occurring, relatively hard, durable resinite polymers derived from the exudates of trees. This resource has been coveted for thousands of years due to its numerous useful properties in industrial processes, beauty, and purported medicinal properties. Labdane diterpenoid based ambers represent the most abundant and important resinites on earth. These resinites are a dwindling, non-renewable natural resource, so a new source of such materials needs to be established. Recent advances in sequencing technologies and biochemical engineering are rapidly accelerating the rate of identifying and assigning function to genes involved in terpenoid biosynthesis, as well as ...


Purification, Crystallization And Preliminary Crystallographic Analysis Of The Sh2 Domain Of Il-2-Inducible T-Cell Kinase, Raji E. Joseph, Nathaniel D. Ginder, Julie A. Hoy, Jay C. Nix, Richard B. Honzatko, Amy H. Andreotti Feb 2011

Purification, Crystallization And Preliminary Crystallographic Analysis Of The Sh2 Domain Of Il-2-Inducible T-Cell Kinase, Raji E. Joseph, Nathaniel D. Ginder, Julie A. Hoy, Jay C. Nix, Richard B. Honzatko, Amy H. Andreotti

Biochemistry, Biophysics and Molecular Biology Publications

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X–Pro imide bond readily undergoes cistrans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cistrans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cistrans proline isomerization in ...


Flight Behaviour Of European Corn Borer Infected With Nosema Pyrausta, D. L. Dorhout, Thomas W. Sappington, L. C. Lewis, Marlin E. Rice Feb 2011

Flight Behaviour Of European Corn Borer Infected With Nosema Pyrausta, D. L. Dorhout, Thomas W. Sappington, L. C. Lewis, Marlin E. Rice

Entomology Publications

The microsporidian Nosema pyrausta Paillot is a common and widespread pathogen of European corn borer, Ostrinia nubilalis (Hübner), in North America and Europe. Nosema negatively affects European corn borer longevity and fecundity. In this study, we used flight mills to examine the effects of Nosema infection on flight activity of unmated males and females at 1 and 3 days after pupal eclosion, taking the level of infection, as measured by number of Nosema spores per mg of tissue, into consideration. Nosema infection had a significant negative effect on distance, duration and speed of the longest uninterrupted flight, as well as ...


Immunoglobulin Structure Exhibits Control Over Cdr Motion, Michael T. Zimmermann, Aris Skliros, Andrzej Kloczkowski, Robert L. Jernigan Jan 2011

Immunoglobulin Structure Exhibits Control Over Cdr Motion, Michael T. Zimmermann, Aris Skliros, Andrzej Kloczkowski, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Motions of the IgG structure are evaluated using normal mode analysis of an elastic network model to detect hinges, the dominance of low frequency modes, and the most important internal motions. One question we seek to answer is whether or not IgG hinge motions facilitate antigen binding. We also evaluate the protein crystal and packing effects on the experimental temperature factors and disorder predictions. We find that the effects of the protein environment on the crystallographic temperature factors may be misleading for evaluating specific functional motions of IgG. The extent of motion of the antigen binding domains is computed to ...


Analysis Of Protein Dynamics Using Local-Dme Calculations, Di Wu, Stephen Smith, Hannah Mahan, Robert L. Jernigan, Zhijun Wu Jan 2011

Analysis Of Protein Dynamics Using Local-Dme Calculations, Di Wu, Stephen Smith, Hannah Mahan, Robert L. Jernigan, Zhijun Wu

Biochemistry, Biophysics and Molecular Biology Publications

Flexibility and dynamics of protein structures are reflected in the B-factors and order parameters obtained experimentally with X-ray crystallography and Nuclear Magnetic Resonance (NMR). Methods such as Normal Mode Analysis (NMA) and Elastic Network Models (ENM) can be used to predict the fluctuations of protein structures for either atomic level or coarse-grained structures. Here, we introduce the Local-Distance Matrix Error (DME), an efficient and simple analytic method to study the fluctuations of protein structures, especially for the ensembles of NMR-determined protein structures. Comparisons with the fluctuations obtained by experiments and other by computations show strong correlations.


Statistical Measures On Residue-Level Protein Structural Properties, Yuanyuan Huang, Steve Bonett, Andrzej Kloczkowski, Robert L. Jernigan, Zhijun Wu Jan 2011

Statistical Measures On Residue-Level Protein Structural Properties, Yuanyuan Huang, Steve Bonett, Andrzej Kloczkowski, Robert L. Jernigan, Zhijun Wu

Biochemistry, Biophysics and Molecular Biology Publications

Background

The atomic-level structural properties of proteins, such as bond lengths, bond angles, and torsion angles, have been well studied and understood based on either chemistry knowledge or statistical analysis. Similar properties on the residue-level, such as the distances between two residues and the angles formed by short sequences of residues, can be equally important for structural analysis and modeling, but these have not been examined and documented on a similar scale. While these properties are difficult to measure experimentally, they can be statistically estimated in meaningful ways based on their distributions in known proteins structures.

Results

Residue-level structural properties ...


Mavens: Motion Analysis And Visualization Of Elastic Networks And Structural Ensembles, Michael T. Zimmermann, Andrzej Kloczkowski, Robert L. Jernigan Jan 2011

Mavens: Motion Analysis And Visualization Of Elastic Networks And Structural Ensembles, Michael T. Zimmermann, Andrzej Kloczkowski, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Background

The ability to generate, visualize, and analyze motions of biomolecules has made a significant impact upon modern biology. Molecular Dynamics has gained substantial use, but remains computationally demanding and difficult to setup for many biologists. Elastic network models (ENMs) are an alternative and have been shown to generate the dominant equilibrium motions of biomolecules quickly and efficiently. These dominant motions have been shown to be functionally relevant and also to indicate the likely direction of conformational changes. Most structures have a small number of dominant motions. Comparing computed motions to the structure's conformational ensemble derived from a collection ...


Exploration Of The Relationship Between Topology And Designability Of Conformations, Sumudu P. Leelananda, Fadi Towfic, Robert L. Jernigan, Andrzej Kloczkowski Jan 2011

Exploration Of The Relationship Between Topology And Designability Of Conformations, Sumudu P. Leelananda, Fadi Towfic, Robert L. Jernigan, Andrzej Kloczkowski

Biochemistry, Biophysics and Molecular Biology Publications

Protein structures are evolutionarily more conserved than sequences, and sequences with very low sequence identity frequently share the same fold. This leads to the concept of protein designability. Some folds are more designable and lots of sequences can assume that fold. Elucidating the relationship between protein sequence and the three-dimensional (3D) structure that the sequence folds into is an important problem in computational structural biology. Lattice models have been utilized in numerous studies to model protein folds and predict the designability of certain folds. In this study, all possible compact conformations within a set of two-dimensional and 3D lattice spaces ...


Crystal Structure Of The Cusba Heavy-Metal Efflux Complex Of Escherichia Coli, Chih-Chia Su, Feng Long, Michael T. Zimmermann, Kanagalaghatta R. Rajashankar, Robert L. Jernigan, Edward W. Yu Jan 2011

Crystal Structure Of The Cusba Heavy-Metal Efflux Complex Of Escherichia Coli, Chih-Chia Su, Feng Long, Michael T. Zimmermann, Kanagalaghatta R. Rajashankar, Robert L. Jernigan, Edward W. Yu

Biochemistry, Biophysics and Molecular Biology Publications

Gram-negative bacteria, such as Escherichia coli, expel toxic chemicals via tripartite efflux pumps spanning both the inner and outer membranes. The three parts are: 1) a membrane fusion protein connecting 2) a substrate-binding inner membrane transporter to 3) an outer membrane-anchored channel in the periplasmic space. A crystallographic model of this tripartite efflux complex has been unavailable simply because co-crystallization of different components of the system has proven to be extremely difficult. We previously described the crystal structures of both the inner membrane transporter CusA1 and membrane fusion protein CusB2 of the CusCBA efflux system3,4 from ...


Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya Jan 2011

Aptamer Functionalized Microcantilever Sensors For Cocaine Detection, Kyungho Kang, Ashish Sachan, Marit Nilsen-Hamilton, Pranav Shrotriya

Biochemistry, Biophysics and Molecular Biology Publications

A cocaine-specific aptamer was used as a receptor molecule in a microcantilever-based surface stress sensor for detection of cocaine molecules. An interferometric technique that relies on measuring differential displacement between two microcantilevers (a sensing/reference pair) was utilized to measure the cocaine/aptamer binding induced surface stress changes. Sensing experiments were performed for different concentrations of cocaine from 25 to 500 μM in order to determine the sensor response as a function of cocaine concentration. In the lower concentration range from 25 to 100 μM, surface stress values increased proportionally to coverage of aptamer/cocaine complexes from 11 to 26 ...


A Nuclear Magnetic Resonance Based Approach To Accurate Functional Annotation Of Putative Enzymes In The Methanogen Methanosarcina Acetivorans, Yihong Chen, Ethel Apolinario, Libuse Brachova, Zvi Kelman, Zhuo Li, Basil Nikolau, Lucas John Showman, Kevin Sowers, John Orban Jan 2011

A Nuclear Magnetic Resonance Based Approach To Accurate Functional Annotation Of Putative Enzymes In The Methanogen Methanosarcina Acetivorans, Yihong Chen, Ethel Apolinario, Libuse Brachova, Zvi Kelman, Zhuo Li, Basil Nikolau, Lucas John Showman, Kevin Sowers, John Orban

Biochemistry, Biophysics and Molecular Biology Publications

Background: Correct annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the ...


Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters Jan 2011

Electrostatic Effects On (Di)Terpene Synthase Product Outcome, Ke Zhou, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to exert little control over the actual reaction, instead largely serving as inert templates. However, recent results highlight stereoelectronic effects exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may drive carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues ...


Cyp99a3: Functional Identification Of A Diterpene Oxidase From The Momilactone Biosynthetic Gene Cluster In Rice, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters Jan 2011

Cyp99a3: Functional Identification Of A Diterpene Oxidase From The Momilactone Biosynthetic Gene Cluster In Rice, Qiang Wang, Matthew L. Hillwig, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant ...


Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson Jan 2011

Structure And Mechanism Of The Diterpene Cyclase Ent-Copalyl Diphosphate Synthase, Mustafa Köksal, Huayou Hu, Robert M. Coates, Reuben J. Peters, David D. Christianson

Biochemistry, Biophysics and Molecular Biology Publications

The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.


Biochemical Characterization Of Bacteriophage T4 Mre11-Rad50 Complex, Timothy J. Herdendorf, Dustin William Albrecht, Stephen J. Benkovic, Scott W, Nelson Jan 2011

Biochemical Characterization Of Bacteriophage T4 Mre11-Rad50 Complex, Timothy J. Herdendorf, Dustin William Albrecht, Stephen J. Benkovic, Scott W, Nelson

Biochemistry, Biophysics and Molecular Biology Publications

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn2+-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence ...


The Epigenetic H3s10 Phosphorylation Mark Is Required For Counteracting Heterochromatic Spreading And Gene Silencing In Drosophila Melanogaster, Chao Wang, Weili Cai, Yeran Li, Huai Deng, Xiaomin Bao, Jack Girton, Jorgen Johansen, Kristen M. Johansen Jan 2011

The Epigenetic H3s10 Phosphorylation Mark Is Required For Counteracting Heterochromatic Spreading And Gene Silencing In Drosophila Melanogaster, Chao Wang, Weili Cai, Yeran Li, Huai Deng, Xiaomin Bao, Jack Girton, Jorgen Johansen, Kristen M. Johansen

Biochemistry, Biophysics and Molecular Biology Publications

The JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein. By transgenically expressing various truncated versions of ...