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Biochemistry, Biophysics and Molecular Biology Publications

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Full-Text Articles in Molecular Biology

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 ...


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 ...


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 ...


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 ...


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 ...


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- ...


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 ...


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 ...


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 ...


A Rapid Fluorometric Assay For The S-Malonyltransacylase Fabd And Other Sulfhydryl Utilizing Enzymes, Aaron M. Marcella, Adam W. Barb Jan 2016

A Rapid Fluorometric Assay For The S-Malonyltransacylase Fabd And Other Sulfhydryl Utilizing Enzymes, Aaron M. Marcella, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes ...


An Encodable Lanthanide Binding Tag With Reduced Size And Flexibility For Measuring Residual Dipolar Couplings And Pseudocontact Shifts In Large Proteins, Adam W. Barb, Ganesh P. Subedi Jan 2016

An Encodable Lanthanide Binding Tag With Reduced Size And Flexibility For Measuring Residual Dipolar Couplings And Pseudocontact Shifts In Large Proteins, Adam W. Barb, Ganesh P. Subedi

Biochemistry, Biophysics and Molecular Biology Publications

Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding ...


Product Rearrangement From Altering A Single Residue In The Rice Syn-Copalyl Diphosphate Synthase, Kevin C. Potter, Meirong Jia, Young J. Hong, Dean Tantillo, Reuben J. Peters Jan 2016

Product Rearrangement From Altering A Single Residue In The Rice Syn-Copalyl Diphosphate Synthase, Kevin C. Potter, Meirong Jia, Young J. Hong, Dean Tantillo, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism.


Labeling Studies Clarify The Committed Step In Bacterial Gibberellin Biosynthesis, Ryan S. Nett, Jeroen S. Dickschat, Reuben J. Peters Jan 2016

Labeling Studies Clarify The Committed Step In Bacterial Gibberellin Biosynthesis, Ryan S. Nett, Jeroen S. Dickschat, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

Bacteria have evolved gibberellin phytohormone biosynthesis independently of plants and fungi. Through 13C-labeling and NMR analysis, the mechanistically unusual “B” ring contraction catalyzed by a cytochrome P450 (CYP114), which is the committed step in gibberellin biosynthesis, was shown to occur via oxidative extrusion of carbon-7 from ent-kaurenoic acid in bacteria. This is identical to the convergently evolved chemical transformation in plants and fungi, suggesting a common semipinacol rearrangement mechanism potentially guided by carbon-4α carboxylate proximity.


Fold-Specific Sequence Scoring Improves Protein Sequence Matching, Sumudu P. Leelananda, Andrzej Kloczkowski, Robert L. Jernigan Jan 2016

Fold-Specific Sequence Scoring Improves Protein Sequence Matching, Sumudu P. Leelananda, Andrzej Kloczkowski, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Background

Sequence matching is extremely important for applications throughout biology, particularly for discovering information such as functional and evolutionary relationships, and also for discriminating between unimportant and disease mutants. At present the functions of a large fraction of genes are unknown; improvements in sequence matching will improve gene annotations. Universal amino acid substitution matrices such as Blosum62 are used to measure sequence similarities and to identify distant homologues, regardless of the structure class. However, such single matrices do not take into account important structural information evident within the different topologies of proteins and treats substitutions within all protein folds identically ...


Distributions Of Experimental Protein Structures On Coarse-Grained Free Energy Landscapes, Kannan Sankar, Jie Liu, Yuan Wang, Robert L. Jernigan Jan 2016

Distributions Of Experimental Protein Structures On Coarse-Grained Free Energy Landscapes, Kannan Sankar, Jie Liu, Yuan Wang, Robert L. Jernigan

Biochemistry, Biophysics and Molecular Biology Publications

Predicting conformational changes of proteins is needed in order to fully comprehend functional mechanisms. With the large number of available structures in sets of related proteins, it is now possible to directly visualize the clusters of conformations and their conformational transitions through the use of principal component analysis. The most striking observation about the distributions of the structures along the principal components is their highly non-uniform distributions. In this work, we use principal component analysis of experimental structures of 50 diverse proteins to extract the most important directions of their motions, sample structures along these directions, and estimate their free ...


Crispr Interference And Priming Varies With Individual Spacer Sequences, Chaoyou Xue, Arun S. Seetharam, Olga Musharova, Konstantin Severinov, Stan J. J. Brouns, Andrew J. Severin, Dipali G. Sashital Dec 2015

Crispr Interference And Priming Varies With Individual Spacer Sequences, Chaoyou Xue, Arun S. Seetharam, Olga Musharova, Konstantin Severinov, Stan J. J. Brouns, Andrew J. Severin, Dipali G. Sashital

Biochemistry, Biophysics and Molecular Biology Publications

CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed ...


High Yield Expression Of Recombinant Human Proteins With The Transient Transfection Of Hek293 Cells In Suspension, Ganesh P. Subedi, Roy W. Johnson, Heather A. Moniz, Kelley W. Moreman, Adam W. Barb Dec 2015

High Yield Expression Of Recombinant Human Proteins With The Transient Transfection Of Hek293 Cells In Suspension, Ganesh P. Subedi, Roy W. Johnson, Heather A. Moniz, Kelley W. Moreman, Adam W. Barb

Biochemistry, Biophysics and Molecular Biology Publications

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein ...


Bridging Between Nma And Elastic Network Models: Preserving All-Atom Accuracy In Coarse-Grained Models, Hyuntae Na, Robert L. Jernigan, Guang Song Oct 2015

Bridging Between Nma And Elastic Network Models: Preserving All-Atom Accuracy In Coarse-Grained Models, Hyuntae Na, Robert L. Jernigan, Guang Song

Biochemistry, Biophysics and Molecular Biology Publications

Dynamics can provide deep insights into the functional mechanisms of proteins and protein complexes. For large protein complexes such as GroEL/GroES with more than 8,000 residues, obtaining a fine-grained all-atom description of its normal mode motions can be computationally prohibitive and is often unnecessary. For this reason, coarse-grained models have been used successfully. However, most existing coarse-grained models use extremely simple potentials to represent the interactions within the coarse-grained structures and as a result, the dynamics obtained for the coarse-grained structures may not always be fully realistic. There is a gap between the quality of the dynamics of ...


Efficient Heterocyclisation By (Di)Terpene Synthases, S. Mafu, K. C. Potter, M. L. Hillwig, S. Schulte, J. Criswell, R. J. Peters Sep 2015

Efficient Heterocyclisation By (Di)Terpene Synthases, S. Mafu, K. C. Potter, M. L. Hillwig, S. Schulte, J. Criswell, R. J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

While cyclic ether forming terpene synthases are known, the basis for such heterocyclisation is unclear. Here it is reported that numerous (di)terpene synthases, particularly including the ancestral ent-kaurene synthase, efficiently produce isomers of manoyl oxide from the stereochemically appropriate substrate. Accordingly, such heterocyclisation is easily accomplished by terpene synthases. Indeed, the use of single residue changes to induce production of the appropriate substrate in the upstream active site leads to efficient bifunctional enzymes producing isomers of manoyl oxide, representing novel enzymatic activity.


Optimization Of Recombinant Expression Enables Discovery Of Novel Cytochrome P450 Activity In Rice Diterpenoid Biosynthesis, Naoki Kitaoka, Yisheng Wu, Meimei Xu, Reuben J. Peters Sep 2015

Optimization Of Recombinant Expression Enables Discovery Of Novel Cytochrome P450 Activity In Rice Diterpenoid Biosynthesis, Naoki Kitaoka, Yisheng Wu, Meimei Xu, Reuben J. Peters

Biochemistry, Biophysics and Molecular Biology Publications

The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here we report that optimization of the ...