Protein Engineering, Design and Selection最新文献_第4页

Chimeric approach for narrowing a membrane-inserting region within human perforin 缩小人穿孔膜插入区域的嵌合方法

Protein Engineering, Design and Selection Pub Date : 2017-02-01 DOI: 10.1093/protein/gzw069

Amy E. Neely, Kimberly A Mandigo, R. Robinson, T. Ness, M. H. Weiland

{"title":"Chimeric approach for narrowing a membrane-inserting region within human perforin","authors":"Amy E. Neely, Kimberly A Mandigo, R. Robinson, T. Ness, M. H. Weiland","doi":"10.1093/protein/gzw069","DOIUrl":"https://doi.org/10.1093/protein/gzw069","url":null,"abstract":"Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved &bgr;-sheet flanked by two clusters of &agr;-helices. Within the CDCs, these helices refold into two transmembrane &bgr;-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79949987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Dual chemistry catalyzed by human acireductone dioxygenase 人酸还原酮双加氧酶催化的双化学反应

Protein Engineering, Design and Selection Pub Date : 2017-01-05 DOI: 10.1093/protein/gzw078

A. Deshpande, T. Pochapsky, G. Petsko, D. Ringe

{"title":"Dual chemistry catalyzed by human acireductone dioxygenase","authors":"A. Deshpande, T. Pochapsky, G. Petsko, D. Ringe","doi":"10.1093/protein/gzw078","DOIUrl":"https://doi.org/10.1093/protein/gzw078","url":null,"abstract":"Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate. This dual chemistry was recently demonstrated in vitro by ARD from Mus musculus (MmARD), providing the first example of a mammalian ARD exhibiting metal-dependent catalysis. We now show that human ARD (HsARD) is also capable of metal-dependent dual chemistry. Recombinant HsARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. As with MmARD, the Fe2+-bound HsARD shows the highest activity and catalyzes on-pathway chemistry, whereas Ni2+, Co2+ or Mn2+ forms catalyze off-pathway chemistry. The thermal stability of the HsARD isozymes is a function of the metal ion identity, with Ni2+-bound HsARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound HsARD being the least stable. As with the bacterial ARD, solution NMR data suggest that HsARD isozymes can have significant structural differences depending upon the metal ion bound.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77928171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10

On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase 碱性pH和辅助因子可用性对大肠杆菌谷氨酸脱羧酶构象和寡聚状态的影响

Protein Engineering, Design and Selection Pub Date : 2017-01-05 DOI: 10.1093/protein/gzw076

Fabio Giovannercole, C. Mérigoux, C. Zamparelli, D. Verzili, G. Grassini, Malcolm Buckle, P. Vachette, D. Biase

{"title":"On the effect of alkaline pH and cofactor availability in the conformational and oligomeric state of Escherichia coli glutamate decarboxylase","authors":"Fabio Giovannercole, C. Mérigoux, C. Zamparelli, D. Verzili, G. Grassini, Malcolm Buckle, P. Vachette, D. Biase","doi":"10.1093/protein/gzw076","DOIUrl":"https://doi.org/10.1093/protein/gzw076","url":null,"abstract":"Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90401219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Generation of camelid VHH bispecific constructs via in-cell intein-mediated protein trans-splicing 通过细胞内蛋白介导的反式剪接产生骆驼类VHH双特异性构建体

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw057

Yukihiko Shibuya, N. Haga, R. Asano, H. Nakazawa, T. Hattori, D. Takeda, Aruto Sugiyama, R. Kurotani, I. Kumagai, M. Umetsu, K. Makabe

引用次数: 8

Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 −) to formate 热毛毛菌甲酸脱氢酶在还原碳酸氢(HCO3−)生成甲酸中具有高活性

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw062

Aşkın Sevinç Aslan, J. Valjakka, Jouni Ruupunen, D. Yıldırım, N. Turner, O. Turunen, Barış Binay

{"title":"Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 −) to formate","authors":"Aşkın Sevinç Aslan, J. Valjakka, Jouni Ruupunen, D. Yıldırım, N. Turner, O. Turunen, Barış Binay","doi":"10.1093/protein/gzw062","DOIUrl":"https://doi.org/10.1093/protein/gzw062","url":null,"abstract":"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. CtFDH showed a higher efficiency in converting HCO3 − to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7–8. However, the high concentrations of HCO3 − reduced the reaction rate. CtFDH was modeled in the presence of HCO3 − showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3 −, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88253575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 29

Allosteric control of antibody-prion recognition through oxidation of a disulfide bond between the CH and CL chains. 通过氧化 CH 链和 CL 链之间的二硫键，对抗体-磷脂识别进行异构控制。

Protein Engineering, Design and Selection Pub Date : 2017-01-01 Epub Date: 2016-11-29 DOI: 10.1093/protein/gzw065

Jun Zhao, Ruth Nussinov, Buyong Ma

{"title":"Allosteric control of antibody-prion recognition through oxidation of a disulfide bond between the CH and CL chains.","authors":"Jun Zhao, Ruth Nussinov, Buyong Ma","doi":"10.1093/protein/gzw065","DOIUrl":"10.1093/protein/gzw065","url":null,"abstract":"<p><p>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. We further identified several negative and positive correlations of motions between the peptide and Fab constant domains, which suggested structural cross-talks between the constant domains and the antigen. The cross-talk was influenced by the inter-chain disulfide bond, which reduced the number of paths between them. Importantly, network analysis of the complex and its bound water molecules observed that those water molecules form an integral part of the Fab/peptide complex network and potential allosteric pathways. On-going work focuses on developing strategies aimed to incorporate these new network communications-including the associated water molecules-toward the grand challenge of antibody design.</p>","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90360671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Stabilization of luciferase from Renilla reniformis using random mutations 随机突变稳定Renilla reniformis荧光素酶

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw056

M. Shigehisa, Norie Amaba, S. Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, F. A. Laksmi, M. Tokunaga, M. Ishibashi

引用次数: 12

Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation 糖工程干扰素的合理设计具有改进聚合状态的类似物:实验验证

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw058

M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh

{"title":"Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation","authors":"M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh","doi":"10.1093/protein/gzw058","DOIUrl":"https://doi.org/10.1093/protein/gzw058","url":null,"abstract":"Recombinant human interferon-&bgr; (rhIFN-&bgr;) 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-&bgr; analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-&bgr; analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-&bgr; (Opt-IFN-&bgr;) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-&bgr;, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-&bgr; had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-&bgr;. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-&bgr; analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74684320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Design and expression of a chimeric vaccine candidate for avian necrotic enteritis 禽坏死性肠炎候选嵌合疫苗的设计与表达

Protein Engineering, Design and Selection Pub Date : 2017-01-01 DOI: 10.1093/protein/gzw060

A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian

{"title":"Design and expression of a chimeric vaccine candidate for avian necrotic enteritis","authors":"A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian","doi":"10.1093/protein/gzw060","DOIUrl":"https://doi.org/10.1093/protein/gzw060","url":null,"abstract":"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. The separate regions were fused together using rigid linkers. Based on a modeled tertiary structure, a proper combination was selected and expressed in a bacterial host (Escherichia coli) and successfully purified. The expression of the chimeric protein was further verified by western blotting. The ability of the immunized serum in recognizing each individual subunit of the chimeric protein was also examined. Circular dichroism was used to evaluate the predicted secondary structure of the chimeric protein. In vitro potency test demonstrated that the serum from a rabbit immunized with the chimeric protein is able to partially neutralize Alpha toxin, hence the construct can potentially be used as a vaccine against C. perfringens.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2017-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82990690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 16

A robust cosolvent-compatible halohydrin dehalogenase by computational library design 基于计算库设计的稳健的助溶剂相容卤代醇脱卤酶

Protein Engineering, Design and Selection Pub Date : 2016-12-19 DOI: 10.1093/protein/gzw068

Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen

{"title":"A robust cosolvent-compatible halohydrin dehalogenase by computational library design","authors":"Hesam Arabnejad, Marco Dal Lago, P. Jekel, Robert J. Floor, A. Thunnissen, A. C. Terwisscha van Scheltinga, Hein J. Wijma, D. Janssen","doi":"10.1093/protein/gzw068","DOIUrl":"https://doi.org/10.1093/protein/gzw068","url":null,"abstract":"To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85654055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 34