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Protein Engineering, Design & Selection: PEDS

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https://www.readbyqxmd.com/read/27903763/enzyme-activation-through-the-utilization-of-intrinsic-dianion-binding-energy
#1
REVIEW
T L Amyes, M M Malabanan, X Zhai, A C Reyes, J P Richard
We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol., 43: , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis...
November 29, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27899437/allosteric-control-of-antibody-prion-recognition-through-oxidation-of-a-disulfide-bond-between-the-ch-and-cl-chains
#2
Jun Zhao, Ruth Nussinov, Buyong Ma
Molecular details of the recognition of disordered antigens by their cognate antibodies have not been studied as extensively as folded protein antigens and much is still unknown. To follow the conformational changes in the antibody and cross-talk between its subunits and with antigens, we performed molecular dynamics (MD) simulations of the complex of Fab and prion-associated peptide in the apo and bound forms. We observed that the inter-chain disulfide bond in constant domains restrains the conformational changes of Fab, especially the loops in the CH1 domain, resulting in inhibition of the cross-talk between Fab subdomains that thereby may prevent prion peptide binding...
November 29, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27887027/expansion-of-the-substrate-range-of-the-gentisate-1-2-dioxygenase-from-corynebacterium-glutamicum-for-the-conversion-of-monohydroxylated-benzoates
#3
Erik Eppinger, Andreas Stolz
The gentisate 1,2-dioxygenases (GDOs) from Corynebacterium glutamicum and various other organisms oxidatively cleave the aromatic nucleus of gentisate (2,5-dihydroxybenzoate), but are not able to convert salicylate (2-hydroxybenzoate). In contrast, the α-proteobacterium Pseudaminobacter salicylatoxidans synthesises an enzyme ('salicylate dioxygenase', SDO) which cleaves gentisate, but also (substituted) salicylate(s). Sequence comparisons showed that the SDO belongs to a group of GDOs mainly originating from Gram-positive bacteria which also include the GDO from C...
November 24, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27887026/chaetomium-thermophilum-formate-dehydrogenase-has-high-activity-in-the-reduction-of-hydrogen-carbonate-hco3-to-formate
#4
Aşkın Sevinç Aslan, Jarkko Valjakka, Jouni Ruupunen, Deniz Yildirim, Nicholas J Turner, Ossi Turunen, Barış Binay
While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO(-)). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3(-)) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3(-) as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays...
November 24, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27887025/design-and-expression-of-a-chimeric-vaccine-candidate-for-avian-necrotic-enteritis
#5
Amin Rostami, Fatemeh Goshadrou, Reza Pilehchian Langroudi, S Zahra Bathaie, Ali Riazi, Jafar Amani, Gholamreza Ahmadian
Necrotic enteritis is an economically important disease of poultry mainly caused by Clostridium perfringens The bacteria release multiple toxins of which NetB, alpha toxin and TpeL have been reported to play important roles in pathogenicity and/or severity of the disease. In this study, the sequence of clostridial toxins NetB, alpha toxin and TpeL were analyzed using bioinformatics tools to determine protein domains with high immunogenicity factor. Several chimeric trivalent proteins consisting of the immunogenic regions of the three toxins were designed and evaluated...
November 24, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27881685/generation-of-camelid-vhh-bispecific-constructs-via-in-cell-intein-mediated-protein-trans-splicing
#6
Yuki Shibuya, Natsuki Haga, Ryutaro Asano, Hikaru Nakazawa, Takamitsu Hattori, Daisuke Takeda, Aruto Sugiyama, Reiko Kurotani, Izumi Kumagai, Mitsuo Umetsu, Koki Makabe
Production of various combinations of bispecific variable domain of heavy chain of heavy chain-only antibody (VHH) constructs to evaluate their therapeutic potential usually requires several gene-engineering steps. Here, we present an alternative method of creating bispecific VHH constructs in vivo through protein trans-splicing (PTS) reaction; this method may reduce the number of gene manipulation steps required. As a proof-of-concept, we constructed a bispecific antibody (bsAb) containing an anti-epidermal growth factor receptor VHH and anti-green fluorescent protein VHH, and we evaluated and confirmed its bispecificity...
November 23, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27881684/conformational-flexibility-of-an-anti-il-13-darpin%C3%A2
#7
Alexey Teplyakov, Thomas J Malia, Galina Obmolova, Steven A Jacobs, Karyn T O'Neil, Gary L Gilliland
Designed ankyrin repeat proteins (DARPin(®)) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution. The DARPin competes for the same epitope as the IL-13 receptor chain 13Rα1 but does not interfere with the binding of the other receptor chain, IL-4Rα...
November 23, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27881683/rational-design-of-glycoengineered-interferon-%C3%AE-analogs-with-improved-aggregation-state-experimental-validation
#8
M Samoudi, Z Minuchehr, S W Harcum, F Tabandeh, N Omid Yeganeh, M Khodabandeh
Recombinant human interferon-β (rhIFN-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-β analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized...
November 23, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27881682/stabilization-of-luciferase-from-renilla-reniformis-using-random-mutations
#9
Megumi Shigehisa, Norie Amaba, Shigeki Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, Fina Amreta Laksmi, Masao Tokunaga, Matsujiro Ishibashi
We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained...
November 23, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27760803/understanding-the-role-of-phosphorylation-in-the-binding-mechanism-of-a-pdz-domain
#10
Angelo Toto, Annalisa Mattei, Per Jemth, Stefano Gianni
The PDZ domain is one of the most common protein-protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL...
October 19, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27744288/direct-determination-of-enzyme-kinetic-parameters-from-single-reactions-using-a-new-progress-curve-analysis-tool
#11
Felix Bäuerle, Agnes Zotter, Gideon Schreiber
With computer-based data-fitting methods becoming a standard tool in biochemistry, progress curve analysis of enzyme kinetics is a feasible, yet seldom used tool. Here we present a versatile Matlab-based tool (PCAT) to analyze catalysis progress curves with three complementary model approaches. The first two models are based on the known closed-form solution for this problem: the first describes the required Lambert W function with an analytical approximation and the second provides a numerical solution of the Lambert W function...
October 15, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27737925/the-integrin-talin-complex-under-force
#12
Jan Neumann, Kay-Eberhard Gottschalk
Integrins are the major transmembrane cellular adhesion receptors. Talin binds to integrins with its head domain and links them to the actin cytoskeleton with its rod domain, acting as the force linkage between the extracellular matrix and the cytoskeleton. It is unknown how forces in different directions affect the integrin-talin complex. We show that small forces applied to the integrin-talin complex breaks a salt bridge between the integrins α- and β-subunit, unlocking the integrin from its resting state...
October 13, 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27737926/engineering-a-targeted-delivery-platform-using-centyrins
#13
Shalom D Goldberg, Rosa M F Cardoso, Tricia Lin, Tracy Spinka-Doms, Donna Klein, Steven A Jacobs, Vadim Dudkin, Gary Gilliland, Karyn T O'Neil
Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27672050/evaluating-the-potential-of-a-loop-extended-scorpion-toxin-like-peptide-as-a-protein-scaffold
#14
Shangfei Zhang, Limei Zhu, Jie Yu, Jun Xu, Bin Gao, Changlin Zhou, Shunyi Zhu
Grafting of exogenous bioactive sites or functional motifs onto structurally stable scaffolds to gain new functions represents an important research direction in protein engineering. Some engineered proteins have been developed into therapeutic drugs. MeuNaTxα-3 (abbreviated as MT-3) is a newly characterized scorpion sodium channel toxin-like peptide isolated from the venom of the scorpion Mesobuthus eupeus, which contains a rigid scaffold highly similar to classical scorpion sodium channel toxins and an extension of eight amino acids in its J-loop region...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27672049/in-vitro-evolution-of-phi29-dna-polymerase-using-isothermal-compartmentalized-self-replication-technique
#15
Tadas Povilaitis, Gediminas Alzbutas, Rasa Sukackaite, Juozas Siurkus, Remigijus Skirgaila
Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27672048/engineering-a-minimal-g-protein-to-facilitate-crystallisation-of-g-protein-coupled-receptors-in-their-active-conformation
#16
Byron Carpenter, Christopher G Tate
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27624308/a-generalized-framework-for-computational-design-and-mutational-scanning-of-t-cell-receptor-binding-interfaces
#17
Timothy P Riley, Cory M Ayres, Lance M Hellman, Nishant K Singh, Michael Cosiano, Jennifer M Cimons, Michael J Anderson, Kurt H Piepenbrink, Brian G Pierce, Zhiping Weng, Brian M Baker
T-cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for cancer where they are the key components of new cellular therapies as well as soluble biologics. Many studies have generated high affinity TCRs in order to enhance sensitivity. Recent outcomes, however, have suggested that fine manipulation of TCR binding, with an emphasis on specificity may be more valuable than large affinity increments. Structure-guided design is ideally suited for this role, and here we studied the generality of structure-guided design as applied to TCRs...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27578886/fusion-of-an-alcohol-dehydrogenase-with-an-aminotransferase-using-a-pas-linker-to-improve-coupled-enzymatic-alcohol-to-amine-conversion
#18
Alexandra Lerchner, Marina Daake, Alexander Jarasch, Arne Skerra
To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT(L417M), as proven by SDS-PAGE and size exclusion chromatography...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27160178/creation-of-zebularine-resistant-human-cytidine-deaminase-mutants-to-enhance-the-chemoprotection-of-hematopoietic-stem-cells
#19
Hongmei Ruan, Songbo Qiu, Brian C Beard, Margaret E Black
Human cytidine deaminase (hCDA) is a biomedically important enzyme able to inactivate cytidine nucleoside analogs such as the antileukemic agent cytosine arabinoside (AraC) and thereby limit antineoplastic efficacy. Potent inhibitors of hCDA have been developed, e.g. zebularine, that when administered in combination with AraC enhance antineoplastic activity. Tandem hematopoietic stem cell (HSC) transplantation and combination chemotherapy (zebularine and AraC) could exhibit robust antineoplastic potency, but AraC-based chemotherapy regimens lead to pronounced myelosuppression due to relatively low hCDA activity in HSCs, and this approach could exacerbate this effect...
December 2016: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/27672106/the-burgeoning-antibody-landscape
#20
James S Huston
No abstract text is available yet for this article.
October 2016: Protein Engineering, Design & Selection: PEDS
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