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    1 OF 1367

    Effect of an Imposed Contact on Secondary Structure in the Denatured State of Yeast Iso-1-Cytochome c.
    Biochemistry 2017 Nov 17. Epub 2017 Nov 17.
    There is considerable evidence that long-range interactions stabilize residual protein structure under denaturing conditions. However, evaluation of the effect of a specific contact on structure in the denatured state has been difficult. Iso-1-cytochrome c variants with a Lys54-->His mutation form a particularly stable His-heme loop in the denatured state, suggestive of loop-induced residual structure. Read More

    Chromophore isomer stabilization is critical to efficient fluorescence in Cyan Fluorescent Proteins.
    Biochemistry 2017 Nov 17. Epub 2017 Nov 17.
    ECFP, the first usable Cyan Fluorescent Protein, was obtained by adapting the tyrosine-based chromophore environment in Green Fluorescent Protein to that of a tryptophan-based one. This 1(st)-generation CFP was superseded by the popular Cerulean, CyPet and SCFP3A that were engineered by rational and random mutagenesis. Yet, the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. Read More

    The sign of NMR chemical shift difference as a determinant of the origin of binding selectivity: Elucidation of the position-dependence of phosphorylation in ligands binding to Scribble PDZ1.
    Biochemistry 2017 Nov 16. Epub 2017 Nov 16.
    The use of NMR chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purposes, amongst others, to determine affinity constants, locate binding surfaces, or differentiate between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Read More

    Differential effects of strand asymmetry on the energetics and structural flexibility of DNA internal loops.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Internal loops within structured nucleic acids disrupt local base-stacking and destabilize neighboring helical domains; however, these structural motifs also expand the conformational and functional capabilities for structured nucleic acids. Variations in size, distribution of loop nucleotides on opposing strands (strand asymmetry), and sequence alter their biophysical properties. Here, the thermodynamics and structural flexibility of poly(T)-rich DNA internal loops were systematically investigated in terms of loop size and strand asymmetry. Read More

    Mutation-Induced Deamidation of Corneal Dystrophy-Related Transforming Growth Factor β-Induced Protein.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Mutations in the transforming growth factor β-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using LC-MS/MS and NMR, we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. Read More

    Tissue Inhibitor of Metalloprotease-2 (TIMP-2): bioprocess development, physicochemical, biochemical and biological characterization of highly expressed recombinant protein.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Tissue Inhibitor of Metalloprotease -2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP-inhibitory activity, . These physiological properties make TIMP-2 an excellent candidate for further pre-clinical development as a biologic therapy of cancer. Read More

    The Regulatory Mechanism of Mycobacterium tuberculosis Phosphoserine Phosphatase SerB2.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Almost all organisms contain the same biosynthetic pathway for the synthesis of l-serine from the glycolytic intermediate, D-3-phosphoglycerate. However, regulation of this pathway varies from organism to organism. Many organisms control the activity of the first enzyme in the pathway, D-3-phosphoglyerate dehydrogenase (PGDH), by feedback inhibition through the interaction of l-serine with the ACT domains within the enzyme. Read More

    Determinants and Expansion of Specificity in a Trichothecene UDP-glucosyltransferase from Oryza sativa.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Family 1 UDP-glycosyltransferases in plants (UGTs) primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, where this is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins on account of its ability to glycosylate DON, nivalenol and hydrolyzed T-2 toxin (HT-2). Read More

    QM/MM Simulations Identify the Ring-Opening Mechanism of Creatininase.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) to creatine via a two-step mechanism: water-addition followed by ring-opening. Water-addition is common to other known cyclic amidohydrolases, but the precise mechanism for ring-opening is still under debate. The proton donor in this step is either His178, or a water molecule bound to one of metal ions, and the roles of His178 and Glu122 are unclear. Read More

    Applications of Oxygenases in the Chemoenzymatic Total Synthesis of Complex Natural Products.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Nature has evolved a diverse range of oxygenases for the modification of secondary metabolites with selectivity profiles that are unmatched by conventional man-made catalysts. In the past two decades, organic chemists have begun to harness the synthetic potential of these biocatalysts to develop efficient chemoenzymatic synthesis of complex natural products. Judicious combination of synthetic and enzymatic transformations in multi-step synthesis can often result in powerful disconnections that compare favorably with contemporary chemical strategies to access the target natural products, while at the same time, presenting opportunities to innovate. Read More

    Characterization of a UGT84 family glycosyltransferase provides new insights into substrate binding and reactivity of galloylglucose ester-forming UGTs.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Galloylated plant specialized metabolites play important roles in plant-environment interactions as well as the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs. Read More

    A Critical Role of Ser26 Hydrogen Bonding in Aβ42 Assembly and Toxicity.
    Biochemistry 2017 Nov 16. Epub 2017 Nov 16.
    Department of Neurology, David Geffen School of Medicine at UCLA , Los Angeles, California 90095, United States.
    Amyloid β-protein (Aβ) assembly is a seminal process in Alzheimer's disease. Elucidating the mechanistic features of this process is thought to be vital for the design and targeting of therapeutic agents. Computational studies of the most pathologic form of Aβ, the 42-residue Aβ42 peptide, have suggested that hydrogen bonding involving Ser26 may be particularly important in organizing a monomer folding nucleus and in subsequent peptide assembly. Read More

    Deoxycholate-Enhanced Shigella Virulence is Regulated by a Rare π-helix in the Type Three Secretion System Tip Protein IpaD.
    Biochemistry 2017 Nov 14. Epub 2017 Nov 14.
    Type three secretion systems (T3SS) are specialized nano-machines that support infection by injecting bacterial proteins directly into host cells. The Shigella T3SS has uniquely evolved to sense environmental levels of the bile salt deoxycholate (DOC) and up-regulate virulence in response to DOC. In this study, we describe a rare i+5 hydrogen bonding secondary structure element (π-helix) within the type three secretion system tip protein IpaD that plays a critical role in DOC-enhanced virulence. Read More

    Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common discovery platform-fragment based drug discovery, two fragments are identified that bind to adjacent sites on a target, and are then linked together using different linkers to identify the linkage for optimum activity. What is not known from these studies is the effects these linkers, that typically contain C, H, and O atoms, have on the properties of the individual fragment. Read More

    X-ray Structure of Catenated Lytic Transglycosylase SltB1.
    Biochemistry 2017 Nov 16. Epub 2017 Nov 16.
    Department of Crystallography and Structural Biology, Institute of Physical Chemistry "Rocasolano", CSIC , 28006 Madrid, Spain.
    Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. Read More

    Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.
    Biochemistry 2017 Nov 17. Epub 2017 Nov 17.
    Department of Biomedical and Molecular Sciences and ‡Protein Function Discovery Group, Queen's University , Kingston, ON K7L 3N6, Canada.
    The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i. Read More

    Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    Plasmodium falciparum parasites are purine auxotrophs that rely exclusively on the salvage of preformed purines from their human hosts to supply the requirement for purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) catalyzes the freely reversible Mg2+-dependent conversion of 6-oxopurine bases to their respective nucleotides and inorganic pyrophosphate. The phosphoribosyl group is derived from 5-phospho--D-ribosyl 1-pyrophosphate (PRPP). Read More

    Constant pH Accelerated Molecular Dynamics Investigation of the pH Regulation Mechanism of Dinoflagellate Luciferase.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    The bioluminescence reaction in dinoflagellates involves the oxidation of an open-chain tetrapyrrole by the enzyme dinoflagellate luciferase (LCF). The activity of LCF is tightly regulated by pH, where the enzyme is essentially inactive at pH ~ 8 and optimally active at pH ~ 6. Little is known about the mechanism of LCF and the structure of the active form of the enzyme, although it has been proposed that several intramolecularly conserved histidine residues in the N-terminal region are important for the pH regulation mechanism. Read More

    Binding and energetics of electron transfer between an artificial four-helix Mn-protein and reaction centers from Rhodobacter sphaeroides.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    The ability of an artificial four-helix bundle Mn-protein, P1, to bind and transfer an electron to photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides was characterized using optical spectroscopy. Upon illumination of reaction centers, an electron is transferred from P, the bacteriochlorophyll dimer, to QA, the primary electron acceptor. The P1 Mn-protein can bind to the reaction center and reduce the oxidized bacteriochlorophyll dimer, P+, with a dissociation constant of 1. Read More

    Resveratrol Exerts Antioxidant Effects by Activating SIRT2 To Deacetylate Prx1.
    Biochemistry 2017 Nov 16. Epub 2017 Nov 16.
    Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences , 15 Datun Road, Chaoyang District, Beijing 100101, China.
    Resveratrol is a promising chemical agent that treats multiple aging-related diseases and improves life span. While reactive oxygen species undoubtedly play ubiquitous roles in the aging process and resveratrol has been shown to be an effective antioxidant, the mechanism through which resveratrol acts against oxidative stress remains unknown. Here we show that resveratrol activates SIRT2 to deacetylate Prx1, leading to an increased H2O2 reduction activity and a decreased cellular H2O2 concentration. Read More

    Selective Binding to mRNA Duplex Regions by Chemically Modified Peptide Nucleic Acids Stimulates Ribosomal Frameshifting.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 637551.
    Minus-one programmed ribosomal frameshifting (-1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon -1 RF, the ribosomal reading frame is shifted from the normal zero frame to the -1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Read More

    Deciphering Nature's Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains.
    Biochemistry 2017 Nov 7. Epub 2017 Nov 7.
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky , Lexington, Kentucky 40536-0596, United States.
    Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(A3aM3SA3bT3) and TioN(AaMNAb). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. Read More

    Functional Characterization of a Condensation Domain that Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens.
    Biochemistry 2017 Nov 7. Epub 2017 Nov 7.
    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of biologically important small molecules. NRPSs can interface with other enzymes to form hybrid biosynthetic systems that expand the structural and functional diversity of their products. The pepteridines are metabolites encoded by an unprecedented pteridine-NRPS-type hybrid biosynthetic gene cluster in Photorhabdus luminescens, but how the distinct enzymatic systems interface to produce these molecules has not been examined at the biochemical level. Read More

    Thermal Shift as an Entropy-Driven Effect.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Bioscience Department, Sygnature Discovery , Nottingham NG1 1GF, U.K.
    Thermal shift assays (TSAs) are among the most commonly used biophysical approaches in drug discovery in both academic and industrial settings. However, the most common interpretation of the data generated by a TSA is purely qualitative, using only the change in melting temperature (ΔTm) as a metric. This has left many questions surrounding the interpretation of the data as well as whether the TSA truly correlates with other assays. Read More

    Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I.
    Biochemistry 2017 Nov 7. Epub 2017 Nov 7.
    Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Read More

    Quantitative and Structural Assessment of Histone Methyllysine Analogue Engagement by Cognate Binding Proteins Reveals Affinity Decrements Relative to Those of Native Counterparts.
    Biochemistry 2017 Nov 10. Epub 2017 Nov 10.
    Department of Chemistry, ‡Department of Molecular Genetics and Cell Biology, and §Department of Biochemistry and Molecular Biology, The University of Chicago , Chicago, Illinois 60637, United States.
    Methyllysine analogues (MLAs), furnished by aminoethylation of engineered cysteine residues, are widely used surrogates of histone methyllysine and are considered to be effective proxies for studying these epigenetic marks in vitro. Here we report the first structure of a trimethyllysine MLA histone in complex with a protein binding partner, quantify the thermodynamic distinctions between MLAs and their native methyllysine counterparts, and demonstrate that these differences can compromise qualitative interpretations of binding at the nucleosome level. Quantitative measurements with two methyllysine binding protein modules reveal substantial affinity losses for the MLA peptides versus the corresponding native methyllysine species in both cases, although the thermodynamic underpinnings are distinct. Read More

    Emerging Functional Differences Between the Synaptotagmin and Ferlin Calcium Sensor Families.
    Biochemistry 2017 Nov 7. Epub 2017 Nov 7.
    The ferlin family of proteins have emerged as multi-C2 domain regulators of calcium triggered membrane fusion and fission events. While initially determined to share many of the features of the synaptotagmins family of calcium sensors, more recent studies have determined that ferlins directly interact with non-neuronal voltage gated calcium channels and nucleate the assembly of membrane trafficking protein complexes, functions that distinguish them from the more well studied synaptotagmins family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmins family. Read More

    Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Department of Chemistry, Boston College , 2609 Beacon Street, Chestnut Hill, Massachusetts 02467, United States.
    Engineered aminoacyl-tRNA synthetase/tRNA pairs that enable site-specific incorporation of noncanonical amino acids (ncAAs) into proteins in living cells have emerged as powerful tools in chemical biology. The Escherichia coli-derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair is a promising candidate for ncAA mutagenesis in mammalian cells, but it has been engineered to charge only a limited set of ncAAs so far. Here we show that two highly polyspecific EcLeuRS mutants can efficiently charge a large array of useful ncAAs into proteins expressed in mammalian cells, while discriminating against the 20 canonical amino acids. Read More

    Combining H/D Exchange Mass Spectrometry and Computational Docking To Derive the Structure of Protein-Protein Complexes.
    Biochemistry 2017 Nov 16. Epub 2017 Nov 16.
    School of Medicine, University of California, San Diego , La Jolla, California 92093, United States.
    Protein-protein interactions are essential for biological function, but structures of protein-protein complexes are difficult to obtain experimentally. To derive the protein complex of the DNA-repair enzyme human uracil-DNA-glycosylase (hUNG) with its protein inhibitor (UGI), we combined rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). Computational docking of the unbound protein structures provides a list of possible three-dimensional models of the complex; DXMS identifies solvent-protected protein residues. Read More

    A sweet embrace: Control of protein-protein interactions by O-linked β-N-acetylglucosamine.
    Biochemistry 2017 Nov 3. Epub 2017 Nov 3.
    O-linked β-N-acetylglucosamine (O-GlcNAc) is a critical post-translational modification (PTM) of thousands of intracellular proteins. Reversible O-GlcNAcylation governs many aspects of cell physiology and is dysregulated in numerous human diseases. Despite this broad pathophysiological significance, major aspects of O-GlcNAc signaling remain poorly understood, including the biochemical mechanisms through which O-GlcNAc transduces information. Read More

    Ex Uno Plura: Differential Labeling of Phospholipid Biosynthetic Pathways with a Single Bioorthogonal Alcohol.
    Biochemistry 2017 Nov 8. Epub 2017 Nov 8.
    Department of Chemistry and Chemical Biology and Weill Institute for Cell and Molecular Biology, Cornell University , Ithaca, New York 14853, United States.
    Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Read More

    Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.
    Biochemistry 2017 Nov 10. Epub 2017 Nov 10.
    Department of Biomolecular Chemistry, Kyoto Prefectural University , Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
    Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Read More

    Biosynthesis of an Opine Metallophore by Pseudomonas aeruginosa.
    Biochemistry 2017 Nov 3;56(45):5967-5971. Epub 2017 Nov 3.
    Department of Molecular Biosciences, University of Kansas , Lawrence, Kansas 66045, United States.
    Bacterial pathogenesis frequently requires metal acquisition by specialized, small-molecule metallophores. We hypothesized that the Gram-negative Pseudomonas aeruginosa encodes the enzymes nicotianamine synthase (NAS) and opine dehydrogenase (ODH), biosynthesizing a new class of opine metallophore, previously characterized only in the unrelated Gram-positive organism Staphylococcus aureus. The identity of this metallophore, herein named pseudopaline, was determined through measurements of binding affinity, the in vitro reconstitution of the biosynthetic pathway to screen potential substrates, and the confirmation of product formation by mass spectrometry. Read More

    Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes.
    Biochemistry 2017 Nov 9. Epub 2017 Nov 9.
    Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University , Sakyo-ku, Kyoto 606-8502, Japan.
    Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial. The present study investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzin-mediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. Read More

    Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A.
    Biochemistry 2017 Nov 15. Epub 2017 Nov 15.
    Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
    Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Read More

    Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.
    Biochemistry 2017 Nov 1;56(45):6051-6060. Epub 2017 Nov 1.
    Marine Science Institute, University of the Philippines , P. Velasquez Street, Diliman, Quezon City 1101, Philippines.
    The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C1-C4, C2-C6, C3-C5. Read More

    Biological Roles of Protein Kinetic Stability.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    Department of Chemistry and Chemical Biology, ‡Center for Biotechnology and Interdisciplinary Studies, and §Biochemistry and Biophysics Graduate Program, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
    A protein's stability may range from nonexistent, as in the case of intrinsically disordered proteins, to very high, as indicated by a protein's resistance to degradation, even under relatively harsh conditions. The stability of this latter group is usually under kinetic control because of a high activation energy for unfolding that virtually traps the protein in a specific conformation, thereby conferring resistance to proteolytic degradation and misfolding aggregation. The usual outcome of kinetic stability is a longer protein half-life. Read More

    Phosphate Promotes the Recovery of Mycobacterium tuberculosis β-Lactamase from Clavulanic Acid Inhibition.
    Biochemistry 2017 Nov 14. Epub 2017 Nov 14.
    Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, Leiden, The Netherlands.
    The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to β-lactam compounds due to its native serine β-lactamase, BlaC. This resistance can be circumvented by administration of a β-lactamase inhibitor. Read More

    Autoantigen La Regulates MicroRNA Processing from Stem-Loop Precursors by Association with DGCR8.
    Biochemistry 2017 Nov 8. Epub 2017 Nov 8.
    Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore , 14 Science Drive 4, Singapore 117543.
    In humans, primary microRNA (pri-miRNA) processing starts from precise cleavage of the stem loop, which is catalyzed by the Drosha-DGCR8 complex. However, the significant inconsistencies in the expression levels among primary, precursor, and mature miRNAs clearly indicate that many other factors may be involved in this regulation. Here, we utilize a newly developed RNA affinity technique to isolate such factors. Read More

    Encapsulation and Controlled Release of Protein Guests by the Bacillus subtilis Lumazine Synthase Capsid.
    Biochemistry 2017 Nov 9. Epub 2017 Nov 9.
    School of Pharmaceutical Science and Technology, Tianjin University , Tianjin 300072, China.
    In Bacillus subtilis, the 60-subunit dodecahedral capsid formed by lumazine synthase (BsLS) acts as a container for trimeric riboflavin synthase (BsRS). To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS, the green fluorescent protein (GFP) was fused to either the last 11 or the last 32 amino acids of BsRS, yielding variant GFP11 or GFP32, respectively. After purification, BsLS capsids that had been co-produced in bacteria with GFP11 and GFP32 are 15- and 6-fold more fluorescent, respectively, than BsLS co-produced with GFP lacking any BsRS fragment, indicating complex formation. Read More

    Comparison of Five Protein Engineering Strategies for Stabilizing an α/β-Hydrolase.
    Biochemistry 2017 Nov 14. Epub 2017 Nov 14.
    Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, University of Minnesota , 1479 Gortner Avenue, Saint Paul, Minnesota 55108, United States.
    A review of the previous stabilization of α/β-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model α/β-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). Read More

    Inhibitory and Non-Inhibitory NH3 Binding at the Water-Oxidizing Manganese Complex of Photosystem II Suggests Possible Sites and a Rearrangement Mode of Substrate Water Molecules.
    Biochemistry 2017 Nov 13. Epub 2017 Nov 13.
    Freie Universität Berlin , Department of Physics, 14195 Berlin, Germany.
    The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH4(+)/NH3 binding. Time-resolved detection of O2 formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH4Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O2 formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV)4 (S3) state. Read More

    Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity.
    Biochemistry 2017 Nov 8. Epub 2017 Nov 8.
    School of Biological Sciences, University of Essex , Wivenhoe Park, Colchester CO4 3SQ, U.K.
    Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. Read More

    Mapping the Small Molecule Interactome by Mass Spectrometry.
    Biochemistry 2017 Nov 10. Epub 2017 Nov 10.
    Department of Chemistry and Chemical Biology, Harvard University , 12 Oxford Street, Cambridge, Massachusetts 02138, United States.
    Mapping small molecule interactions throughout the proteome provides the critical structural basis for functional analysis of their impact on biochemistry. However, translation of mass spectrometry-based proteomics methods to directly profile the interaction between a small molecule and the whole proteome is challenging because of the substoichiometric nature of many interactions, the diversity of covalent and noncovalent interactions involved, and the subsequent computational complexity associated with their spectral assignment. Recent advances in chemical proteomics have begun fill this gap to provide a structural basis for the breadth of small molecule-protein interactions in the whole proteome. Read More

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