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

Gene Pyramids And The Balancing Act Of Keeping Pests At Bay, Gustavo C. Macintosh Sep 2019

Gene Pyramids And The Balancing Act Of Keeping Pests At Bay, Gustavo C. Macintosh

Biochemistry, Biophysics and Molecular Biology Publications

Pyramiding R genes is a common strategy used by breeders to enhance resistance and increase durability of resistance in crops. However, the molecular mechanisms that mediate R gene interactions are not known. Kamphuis et al. (2019) analyzed Medicago truncatula plants carrying two genes that confer resistance to bluegreen aphids. They identified a potential phytohormone crosstalk triggered by the combined R gene action in response to aphid feeding that enhances resistance and minimizes R gene-associated fitness costs to the plant.


Aptamers For Diagnostics With Applications For Infectious Diseases, Muslum Ilgu, Rezzan Fazlioglu, Meric Ozturk, Yasemin Ozsurekci, Marit Nilsen-Hamilton Apr 2019

Aptamers For Diagnostics With Applications For Infectious Diseases, Muslum Ilgu, Rezzan Fazlioglu, Meric Ozturk, Yasemin Ozsurekci, Marit Nilsen-Hamilton

Biochemistry, Biophysics and Molecular Biology Publications

Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and ...


Entropy, Fluctuations, And Disordered Proteins, Eshel Faraggi, A. Keith Dunker, Robert L. Jernigan, Andrzej Kloczkowski Jan 2019

Entropy, Fluctuations, And Disordered Proteins, Eshel Faraggi, A. Keith Dunker, Robert L. Jernigan, Andrzej Kloczkowski

Biochemistry, Biophysics and Molecular Biology Publications

Entropy should directly reflect the extent of disorder in proteins. By clustering structurally related proteins and studying the multiple-sequence-alignment of the sequences of these clusters, we were able to link between sequence, structure, and disorder information. We introduced several parameters as measures of fluctuations at a given MSA site and used these as representative of the sequence and structure entropy at that site. In general, we found a tendency for negative correlations between disorder and structure, and significant positive correlations between disorder and the fluctuations in the system. We also found evidence for residue-type conservation for those residues proximate to ...


Switching On A Nontraditional Enzymatic Base—Deprotonation By Serine In The Ent-Kaurene Synthase From Bradyrhizobium Japonicum, Meirong Jia, Yue Zhang, Justin B. Siegel, Dean J. Tantillo, Reuben J. Peters Jan 2019

Switching On A Nontraditional Enzymatic Base—Deprotonation By Serine In The Ent-Kaurene Synthase From Bradyrhizobium Japonicum, Meirong Jia, Yue Zhang, Justin B. Siegel, Dean J. Tantillo, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Terpene synthases often catalyze complex carbocation cascade reactions. It has been previously shown that single-residue switches involving replacement of a key aliphatic residue with serine or threonine can “short-circuit” such reactions that are presumed to act indirectly via dipole stabilization of intermediate carbocations. Here a similar switch was found in the structurally characterized ent-kaurene synthase from Bradyrhizobium japonicum. Application of a recently developed computational approach to terpene synthases, TerDockin, surprisingly indicates direct action of the introduced serine hydroxyl as a catalytic base. Notably, this model suggests an alternative interpretation of previous results and potential routes toward reengineering terpene synthase ...


Evidence For A Role Of Spindle Matrix Formation In Cell Cycle Progression By Antibody Perturbation, Changfu Yao, Chao Wang, Yeran Li, Michael Zavortink, Vincent Archambault, Jack Girton, Kristen M. Johansen, Jorgen Johansen Nov 2018

Evidence For A Role Of Spindle Matrix Formation In Cell Cycle Progression By Antibody Perturbation, Changfu Yao, Chao Wang, Yeran Li, Michael Zavortink, Vincent Archambault, Jack Girton, Kristen M. Johansen, Jorgen Johansen

Biochemistry, Biophysics and Molecular Biology Publications

In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown. Here using a live imaging approach we provide evidence that a number of key cell cycle proteins such as Cyclin B, Polo, and Ran co-localize with the spindle matrix during mitosis. Furthermore, prevention of spindle matrix formation ...


Arginine In The Farm And Sarm: A Role In Chain-Length Determination For Arginine In The Aspartate-Rich Motifs Of Isoprenyl Diphosphate Synthases From Mycobacterium Tuberculosis, Raimund Nagel, Jill A. Thomas, Faith A. Adekunle, Francis M. Mann, Reuben J. Peters Oct 2018

Arginine In The Farm And Sarm: A Role In Chain-Length Determination For Arginine In The Aspartate-Rich Motifs Of Isoprenyl Diphosphate Synthases From Mycobacterium Tuberculosis, Raimund Nagel, Jill A. Thomas, Faith A. Adekunle, Francis M. Mann, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Isoprenyl chains are found in many important metabolites. These are derived from precursors of the appropriate length produced by isoprenyl diphosphate synthases (IDSs). The human pathogen Mycobacterium tuberculosis makes various isoprenoids/terpenoids, with important roles in their biosynthesis played by two closely related IDSs, encoded by grcC1 (Rv0562) and grcC2 (Rv0989c), with Rv0989c generating the 10-carbon precursor (E)-geranyl diphosphate (GPP), and Rv0562 the 20-carbon precursor (E,E,E)-geranylgeranyl diphosphate (GGPP). Intriguingly, while Rv0562 contains the prototypical trans-IDS first and second aspartate-rich (DDxxD) motifs (FARM and SARM, respectively), Rv0989c uniquely contains arginine in place of the second Asp in ...


Continuous Interdomain Orientation Distributions Reveal Components Of Binding Thermodynamics, Yang Qi, Jeffrey W. Martin, Adam W. Barb, Francois Thelot, Anthony Yan, Bruce R. Donald, Terrence G. Oas Sep 2018

Continuous Interdomain Orientation Distributions Reveal Components Of Binding Thermodynamics, Yang Qi, Jeffrey W. Martin, Adam W. Barb, Francois Thelot, Anthony Yan, Bruce R. Donald, Terrence G. Oas

Biochemistry, Biophysics and Molecular Biology Publications

The flexibility of biological macromolecules is an important structural determinant of function. Unfortunately, the correlations between different motional modes are poorly captured by discrete ensemble representations. Here, we present new ways to both represent and visualize correlated interdomain motions. Interdomain motions are determined directly from residual dipolar couplings, represented as a continuous conformational distribution, and visualized using the disk-on-sphere representation. Using the disk-on-sphere representation, features of interdomain motions, including correlations, are intuitively visualized. The representation works especially well for multidomain systems with broad conformational distributions.This analysis also can be extended to multiple probability density modes, using a Bingham mixture ...


A 2′Fy-Rna Motif Defines An Aptamer For Ebolavirus Secreted Protein, Shambhavi Shubham, Jan Hoinka, Soma Banerjee, Emma Swanson, Jacob A. Dillard, Nicholas J. Lennemann, Teresa M. Przytycka, Wendy Maury, Marit Nilsen-Hamilton Aug 2018

A 2′Fy-Rna Motif Defines An Aptamer For Ebolavirus Secreted Protein, Shambhavi Shubham, Jan Hoinka, Soma Banerjee, Emma Swanson, Jacob A. Dillard, Nicholas J. Lennemann, Teresa M. Przytycka, Wendy Maury, Marit Nilsen-Hamilton

Biochemistry, Biophysics and Molecular Biology Publications

With properties such as stability to long-term storage and amenability to repetitive use, nucleic acid aptamers are compatible with many sensing/transducing platforms intended for use in remote locations. Sensors with these properties are important for quickly identifying ebolavirus outbreaks, which frequently start in locations that lack sophisticated equipment. Soluble glycoprotein (sGP), an excellent biomarker for ebolaviruses, is produced from the same gene as the ebolavirus glycoprotein GP1,2 that decorates the surface of the viral particle and is secreted in abundance into the blood stream even during the early stages of infection. Here, we report the selection and properties ...


Protein Dynamic Communities From Elastic Network Models Align Closely To The Communities Defined By Molecular Dynamics, Sambit Kumar Mishra, Robert L. Jernigan Jun 2018

Protein Dynamic Communities From Elastic Network Models Align Closely To The Communities Defined By Molecular Dynamics, Sambit Kumar Mishra, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Dynamic communities in proteins comprise the cohesive structural units that individually exhibit rigid body motions. These can correspond to structural domains, but are usually smaller parts that move with respect to one another in a protein’s internal motions, key to its functional dynamics. Previous studies emphasized their importance to understand the nature of ligand-induced allosteric regulation. These studies reported that mutations to key community residues can hinder transmission of allosteric signals among the communities. Usually molecular dynamic (MD) simulations (~ 100 ns or longer) have been used to identify the communities—a demanding task for larger proteins. In the present ...


Probing The Specificity Of Cyp112 In Bacterial Gibberellin Biosynthesis, Raimund Nagel, Reuben J. Peters Jun 2018

Probing The Specificity Of Cyp112 In Bacterial Gibberellin Biosynthesis, Raimund Nagel, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and γ-lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here the substrate specificity of the isozyme from Erwinia tracheiphila, EtCYP112, was probed via UV-Vis spectral binding studies and activity assays with ...


Direct Production Of Dihydroxylated Sesquiterpenoids By A Maize Terpene Synthase, Jin Liang, Jiang Liu, Reid Brown, Meirong Jia, Ke Zhou, Reuben J. Peters, Qiang Wang Jun 2018

Direct Production Of Dihydroxylated Sesquiterpenoids By A Maize Terpene Synthase, Jin Liang, Jiang Liu, Reid Brown, Meirong Jia, Ke Zhou, Reuben J. Peters, Qiang Wang

Biochemistry, Biophysics and Molecular Biology Publications

The astounding structural and biological diversity of the large class of terpenoid natural products are imparted by both their complex hydrocarbon backbones and further elaboration by the addition of multiple hydroxyl groups, which provide both solubility and specific binding properties. While the role of terpene synthases in generating hydrocarbons with complex backbones is well known, these also are known to generate (singly) hydroxylated products by the addition of water prior to terminating deprotonation. Here a maize sesquiterpene synthase was unexpectedly found to generate dually hydroxylated products directly from (E,E)-farnesyl diphosphate, primarily eudesmane-2,11-diol, along with two closely related ...


A Dendritic Guidance Receptor Complex Brings Together Distinct Actin Regulators To Drive Efficient F-Actin Assembly And Branching, Wei Zhou, Xintong Dong, Timothy R. Broederdorf, Ao Shen, Daniel A. Kramer, Rebecca Shi, Xing Liang, David M. Miller Iii, Yang K. Xiang, Ryohei Yasuda, Baoyu Chen, Kang Shen May 2018

A Dendritic Guidance Receptor Complex Brings Together Distinct Actin Regulators To Drive Efficient F-Actin Assembly And Branching, Wei Zhou, Xintong Dong, Timothy R. Broederdorf, Ao Shen, Daniel A. Kramer, Rebecca Shi, Xing Liang, David M. Miller Iii, Yang K. Xiang, Ryohei Yasuda, Baoyu Chen, Kang Shen

Biochemistry, Biophysics and Molecular Biology Publications

Proper morphogenesis of dendrites plays a fundamental role in the establishment of neural circuits. The molecular mechanism by which dendrites grow highly complex branches is not well understood. Here, using the C. elegans PVD neuron, we demonstrate that high-order dendritic branching requires actin polymerization driven by coordinated interactions between two membrane proteins, DMA-1 and HPO-30, and with their cytoplasmic interactors, the RacGEF TIAM-1 and the actin nucleation promotion factor WAVE Regulatory Complex (WRC). The dendrite branching receptor DMA-1 directly binds to the PDZ domain of TIAM-1, while the claudin-like protein HPO-30 directly interacts with the WRC. On dendrites, DMA-1 and ...


Restricted Processing Of Cd16a/Fc Γ Receptor Iiia N-Glycans From Primary Human Nk Cells Impacts Structure And Function, Kashyap R. Patel, Jacob T. Roberts, Ganesh P. Subedi, Adam W. Barb Mar 2018

Restricted Processing Of Cd16a/Fc Γ Receptor Iiia N-Glycans From Primary Human Nk Cells Impacts Structure And Function, Kashyap R. Patel, Jacob T. Roberts, Ganesh P. Subedi, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody–CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan ...


Seqstruct : A New Amino Acid Similarity Matrix Based On Sequence Correlations And Structural Contacts Yields Sequence-Structure Congruence, Kejue Jia, Robert L. Jernigan Feb 2018

Seqstruct : A New Amino Acid Similarity Matrix Based On Sequence Correlations And Structural Contacts Yields Sequence-Structure Congruence, Kejue Jia, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Protein sequence matching does not properly account for some well-known features of protein structures: surface residues being more variable than core residues, the high packing densities in globular proteins, and does not yield good matches of sequences of many proteins known to be close structural relatives. There are now abundant protein sequences and structures to enable major improvements to sequence matching. Here, we utilize structural frameworks to mount the observed correlated sequences to identify the most important correlated parts. The rationale is that protein structures provide the important physical framework for improving sequence matching. Combining the sequence and structure data ...


Mouse Igg2c Fc Loop Residues Promote Greater Receptor-Binding Affinity Than Mouse Igg2b Or Human Igg1, Daniel J. Falconer, Adam W. Barb Feb 2018

Mouse Igg2c Fc Loop Residues Promote Greater Receptor-Binding Affinity Than Mouse Igg2b Or Human Igg1, Daniel J. Falconer, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in ...


Comparisons Of Protein Dynamics From Experimental Structure Ensembles, Molecular Dynamics Ensembles, And Coarse-Grained Elastic Network Models, Kannan Sankar, Sambit K. Mishra, Robert L. Jernigan Jan 2018

Comparisons Of Protein Dynamics From Experimental Structure Ensembles, Molecular Dynamics Ensembles, And Coarse-Grained Elastic Network Models, Kannan Sankar, Sambit K. Mishra, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Predicting protein motions is important for bridging the gap between protein structure and function. With growing numbers of structures of the same, or closely related proteins becoming available, it is now possible to understand more about the intrinsic dynamics of a protein with principal component analysis (PCA) of the motions apparent within ensembles of experimental structures. In this paper, we compare the motions extracted from experimental ensembles of 50 different proteins with the modes of motion predicted by several types of coarse-grained elastic network models (ENMs) which additionally take into account more details of either the protein geometry or the ...


Biosynthesis Of Diterpenoids In Tripterygium Adventitious Root Cultures, Fainmarinat S. Inabuy, Justin T. Fischedick, Iris Lange, Michael Hartmann, Narayanan Srividya, Amber N. Parrish, Meimei Xu, Reuben J. Peters, B. Markus Lange Sep 2017

Biosynthesis Of Diterpenoids In Tripterygium Adventitious Root Cultures, Fainmarinat S. Inabuy, Justin T. Fischedick, Iris Lange, Michael Hartmann, Narayanan Srividya, Amber N. Parrish, Meimei Xu, Reuben J. Peters, B. Markus Lange

Biochemistry, Biophysics and Molecular Biology Publications

Adventitious root cultures were developed from Tripterygium regelii Sprague & Takeda and growth conditions optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f and four to the TPS-b subfamily. These genes were characterized by heterologous expression in a modular metabolic engineering ...


Carbohydrate–Polypeptide Contacts In The Antibody Receptor Cd16a Identified Through Solution Nmr Spectroscopy, Ganesh P. Subedi, Daniel J. Falconer, Adam W. Barb Jun 2017

Carbohydrate–Polypeptide Contacts In The Antibody Receptor Cd16a Identified Through Solution Nmr Spectroscopy, Ganesh P. Subedi, Daniel J. Falconer, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

Asparagine-linked carbohydrates (N-glycans) are a common modification of eukaryotic proteins that confer multiple properties including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy to identify N-glycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc γ receptor IIIA). Human embryonic kidney 293S cells expressed CD16A with [13CU]-labeled N-glycans using standard protein expression techniques and medium supplemented with 3 g/L [13CU]-glucose. Anomeric resonances on the protein-linked N-acetylglucosamine residue at the reducing end of the glycan are particularly well suited ...


Investigating The Phylogenetic Range Of Gibberellin Biosynthesis In Bacteria, Raimund Nagel, Reuben J. Peters Apr 2017

Investigating The Phylogenetic Range Of Gibberellin Biosynthesis In Bacteria, Raimund Nagel, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Certain plant-associated microbes can produce gibberellin (GA) phytohormones, as first described for the rice fungal pathogen Gibberella fujikuroi and, more recently, for bacteria, including several rhizobia and the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola. The relevant enzymes are encoded by a biosynthetic operon that exhibits both a greater phylogenetic range and scattered distribution among plant-associated bacteria. Here, the phylogenetic distribution of this operon was investigated. To demonstrate conserved functionality, the enzymes encoded by the disparate operon from X. translucens pv. translucens, along with those from the most divergent example, found in Erwinia tracheiphila, were biochemically characterized. In both of ...


A Pair Of Residues That Interactively Affect Diterpene Synthase Product Outcome, Meirong Jia, Samuel Tufts, Samuel Schulte, Reuben J. Peters Jan 2017

A Pair Of Residues That Interactively Affect Diterpene Synthase Product Outcome, Meirong Jia, Samuel Tufts, Samuel Schulte, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

The labdane-related diterpenoids (LRDs) are an important superfamily of natural products whose structural diversity critically depends on the hydrocarbon skeletal structures generated, in large part, by class I diterpene synthases. In the plant kingdom, where the LRDs are predominantly found, the relevant class I diterpene synthases are clearly derived from the ent-kaurene synthase (KS) required in all land plants for phytohormone biosynthesis and, hence, are often termed KS-like (KSL). Previous work, initiated by the distinct function of two alleles of a KSL from rice, OsKSL5, identified a single residue switch with a profound effect on not only OsKSL5 product ...


H2av Facilitates H3s10 Phosphorylation But Is Not Required For Heat Shock-Induced Chromatin Decondensation Or Transcriptional Elongation, Yeran Li, Chao Wang, Weili Cai, Saheli Sengupta, Michael Zavortink, Huai Deng, Jack Girton, Jorgen Johansen, Kristen M. Johansen Jan 2017

H2av Facilitates H3s10 Phosphorylation But Is Not Required For Heat Shock-Induced Chromatin Decondensation Or Transcriptional Elongation, Yeran Li, Chao Wang, Weili Cai, Saheli Sengupta, Michael Zavortink, Huai Deng, Jack Girton, Jorgen Johansen, Kristen M. Johansen

Biochemistry, Biophysics and Molecular Biology Publications

A model has been proposed in which JIL-1 kinase-mediated H3S10 and H2Av phosphorylation is required for transcriptional elongation and heat shock-induced chromatin decondensation. However, here we show that although H3S10 phosphorylation is indeed compromised in the H2Av null mutant, chromatin decondensation at heat shock loci is unaffected in the absence of JIL-1 as well as of H2Av and that there is no discernable decrease in the elongating form of RNA polymerase II in either mutant. Furthermore, mRNA for the major heat shock protein Hsp70 is transcribed at robust levels in both H2Avand JIL-1 null mutants. Using a different chromatin ...


Cis Or Trans With Class Ii Diterpene Cyclases, Meirong Jia, Reuben J. Peters Jan 2017

Cis Or Trans With Class Ii Diterpene Cyclases, Meirong Jia, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Isoprenoid precursors readily undergo (poly)cyclization in electrophilic reaction cascades, presumably as internal addition of the carbon–carbon double-bonds from neighboring isoprenyl repeats readily forms relatively stable cyclohexyl tertiary carbocation intermediates. This hypothesis is agnostic regarding alkene configuration (i.e., Z or E). Consistent with this, here it is shown that certain class II diterpene cyclases, which normally convert (E,E,E)-geranylgeranyl diphosphate to 13E-trans-decalin bicycles, will also act upon (Z,Z,Z)-nerylneryl diphosphate, producing novel 13Z-cis-decalin bicycles instead.


Rac1 Gtpase Activates The Wave Regulatory Complex Through Two Distinct Binding Sites, Baoyu Chen, Hui-Ting Chou, Chad A. Brautigam, Wenmin Xing, Sheng Yang, Lisa Henry, Lynda K. Doolittle, Thomas Walz, Michael K. Rosen Jan 2017

Rac1 Gtpase Activates The Wave Regulatory Complex Through Two Distinct Binding Sites, Baoyu Chen, Hui-Ting Chou, Chad A. Brautigam, Wenmin Xing, Sheng Yang, Lisa Henry, Lynda K. Doolittle, Thomas Walz, Michael K. Rosen

Biochemistry, Biophysics and Molecular Biology Publications

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC ...


Probing Labdane-Related Diterpenoid Biosynthesis In The Fungal Genus Aspergillus, Meimei Xu, Matthew L. Hillwig, Mollie S. Tiernan, Reuben J. Peters Jan 2017

Probing Labdane-Related Diterpenoid Biosynthesis In The Fungal Genus Aspergillus, Meimei Xu, Matthew L. Hillwig, Mollie S. Tiernan, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid ...


Direct Characterization Of The Maize Starch Synthase Iia Product Shows Maltodextrin Elongation Occurs At The Non-Reducing End, Mark E. Larson, Daniel J. Falconer, Alan M. Myers, Adam W. Barb Nov 2016

Direct Characterization Of The Maize Starch Synthase Iia Product Shows Maltodextrin Elongation Occurs At The Non-Reducing End, Mark E. Larson, Daniel J. Falconer, Alan M. Myers, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

A comprehensive description of starch biosynthesis and granule assembly remains undefined despite the central nature of starch as an energy storage molecule in plants and as a fundamental calorie source for many animals. Multiple theories regarding the starch synthase (SS)-catalyzed assembly of (α1–4)-linked D-glucose molecules into maltodextrins generally agree that elongation occurs at the non-reducing terminus based on the degradation of radiolabeled maltodextrins, although recent reports challenge this hypothesis. Surprisingly, a direct analysis of the SS catalytic product has not been reported, to our knowledge. We expressed and characterized recombinant Zea mays SSIIa and prepared pure ADP- ...


Digitor/Dasciz Has Multiple Roles In Drosophila Development, Saheli Sengupta, Uttama Rath, Changfu Yao, Michael Zavortink, Chao Wang, Jack Girton, Kristen M. Johansen, Jorgen Johansen Nov 2016

Digitor/Dasciz Has Multiple Roles In Drosophila Development, Saheli Sengupta, Uttama Rath, Changfu Yao, Michael Zavortink, Chao Wang, Jack Girton, Kristen M. Johansen, Jorgen Johansen

Biochemistry, Biophysics and Molecular Biology Publications

In this study we provide evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor/dASCIZ. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. We also confirm a previously documented function of Digitor/dASCIZ as a regulator of Dynein light chain/Cut up expression. Using transgenic expression of a mCitrine-labeled Digitor construct, we show that Digitor/dASCIZ is a nuclear protein that is localized to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during ...


Extending A Single Residue Switch For Abbreviating Catalysis In Plant Ent-Kaurene Synthases, Meirong Jia, Reuben J. Peters Nov 2016

Extending A Single Residue Switch For Abbreviating Catalysis In Plant Ent-Kaurene Synthases, Meirong Jia, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Production of ent-kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s) present in all land plants (i.e., embryophyta). The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP) and the subsequently acting class I diterpene synthase that produces ent-kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases and ent-kaurene synthases (KSs ...


Identification Of A Dolabellane Type Diterpene Synthase And Other Root-Expressed Diterpene Synthases In Arabidopsis, Qiang Wang, Meirong Jia, Jung-Hyun Huh, Andrew Muchlinski, Reuben J. Peters, Dorothea Tholl Nov 2016

Identification Of A Dolabellane Type Diterpene Synthase And Other Root-Expressed Diterpene Synthases In Arabidopsis, Qiang Wang, Meirong Jia, Jung-Hyun Huh, Andrew Muchlinski, Reuben J. Peters, Dorothea Tholl

Biochemistry, Biophysics and Molecular Biology Publications

Arabidopsis thaliana maintains a complex metabolism for the production of secondary or specialized metabolites. Such metabolites include volatile and semivolatile terpenes, which have been associated with direct and indirect defensive activities in flowers and leaves. In comparison, the structural diversity and function of terpenes in Arabidopsis roots has remained largely unexplored despite a substantial number of root-expressed genes in the Arabidopsis terpene synthase (TPS) gene family. We show that five root-expressed TPSs of an expanded subfamily-a type clade in the Arabidopsis TPS family function as class I diterpene synthases that predominantly convert geranylgeranyl diphosphate (GGPP) to different semi-volatile diterpene products ...


Dynamic Allostery Mediated By A Conserved Tryptophan In The Tec Family Kinases, Nikita Chopra, Thomas E. Wales, Raji E. Joseph, Scott E. Boyken, John R. Engen, Robert L. Jernigan, Amy H. Andreotti Mar 2016

Dynamic Allostery Mediated By A Conserved Tryptophan In The Tec Family Kinases, Nikita Chopra, Thomas E. Wales, Raji E. Joseph, Scott E. Boyken, John R. Engen, Robert L. Jernigan, Amy H. Andreotti

Biochemistry, Biophysics and Molecular Biology Publications

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how ...


Blocking Deprotonation With Retention Of Aromaticity In A Plant Ent-Copalyl Diphosphate Synthase Leads To Product Rearrangement, Kevin C. Potter, Jiachen Zi, Young J. Hong, Samuel Schulte, Brandi Malchow, Dean J. Tantillo, Reuben J. Peters Jan 2016

Blocking Deprotonation With Retention Of Aromaticity In A Plant Ent-Copalyl Diphosphate Synthase Leads To Product Rearrangement, Kevin C. Potter, Jiachen Zi, Young J. Hong, Samuel Schulte, Brandi Malchow, Dean J. Tantillo, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Biosynthesis of the labdane-related diterpenoids, a large superfamily of over 7,000 natural products, is characterized by the use of class II diterpene cyclases.[1] The vast majority of these biocatalysts are exclusively found in plants, stemming from repeated evolutionary diversification of the ent-copalyl diphosphate synthase (CPS) required for gibberellin phytohormone biosynthesis – i.e., via gene duplication and neofunctionalization.[2] As implied by this evolutionary scenario, these enzymes must exhibit catalytic plasticity that enables new products to readily arise. Consistent with this hypothesis, we have shown that substitution of smaller residues for the catalytic base group can lead to the ...