Protein Engineering, Design and Selection最新文献_第5页

A theoretical study on the reactivity of the Mo/Cu-containing carbon monoxide dehydrogenase with dihydrogen 含Mo/ cu一氧化碳脱氢酶与二氢反应性的理论研究

Protein Engineering, Design and Selection Pub Date : 2016-12-19 DOI: 10.1093/protein/gzw071

R. Breglia, M. Bruschi, U. Cosentino, L. De Gioia, C. Greco, Toshiko Miyake, G. Moro

引用次数: 4

Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates 谷氨酸棒状杆菌中龙胆酸1,2-双加氧酶底物范围的扩大，用于单羟基苯甲酸盐的转化

Protein Engineering, Design and Selection Pub Date : 2016-11-24 DOI: 10.1093/protein/gzw061

E. Eppinger, A. Stolz

{"title":"Expansion of the substrate range of the gentisate 1,2-dioxygenase from Corynebacterium glutamicum for the conversion of monohydroxylated benzoates","authors":"E. Eppinger, A. Stolz","doi":"10.1093/protein/gzw061","DOIUrl":"https://doi.org/10.1093/protein/gzw061","url":null,"abstract":"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 &agr;-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. glutamicum ATCC 13032. The combination of sequence comparisons with previously performed structural and mutational analyses of the SDO allowed to identify an amino acid residue (Ala112) which might prevent the oxidation of (substituted) salicylate(s) by the GDO from C. glutamicum. Therefore, the relevant mutation (Ala→Gly) was introduced into the GDO from C. glutamicum. The GDO variant obtained gained the ability to oxidise salicylate and several other monohydroxylated substrates. In order to screen a broader range of enzyme variants a chromogenic assay was developed which allowed the detection of bacterial colonies converting salicylate. The applicability of this test system was proven by screening a set of GDO variants obtained by saturation mutagenesis at different positions. This demonstrated that also GDO variants carrying the mutations Ala112→Ser, Ala112→Ile and Ala112→Asp converted salicylate.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85321721","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

Conformational flexibility of an anti-IL-13 DARPin† 抗il -13 DARPin†的构象柔韧性

Protein Engineering, Design and Selection Pub Date : 2016-11-23 DOI: 10.1093/protein/gzw059

A. Teplyakov, T. Malia, G. Obmolova, S. Jacobs, K. O'Neil, G. Gilliland

{"title":"Conformational flexibility of an anti-IL-13 DARPin†","authors":"A. Teplyakov, T. Malia, G. Obmolova, S. Jacobs, K. O'Neil, G. Gilliland","doi":"10.1093/protein/gzw059","DOIUrl":"https://doi.org/10.1093/protein/gzw059","url":null,"abstract":"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 13R&agr;1 but does not interfere with the binding of the other receptor chain, IL-4R&agr;. Analysis of multiple copies of the DARPin molecule in the crystal indicates the conformational instability in the N-terminal cap that was predicted from molecular dynamics simulations. Comparison of the DARPin structures in the free state and in complex with IL-13 reveals a concerted movement of the ankyrin repeats upon binding resulted in the opening of the binding site. The induced-fit mode of binding employed by DARPin 6G9 is very unusual for DARPins since they were designed as particularly stable and rigid molecules. This finding shows that DARPins can operate by various binding mechanisms and suggests that some flexibility in the scaffold may be an advantage.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76934647","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: 9

Understanding the role of phosphorylation in the binding mechanism of a PDZ domain 了解磷酸化在PDZ结构域结合机制中的作用

Protein Engineering, Design and Selection Pub Date : 2016-10-19 DOI: 10.1093/protein/gzw055

A. Toto, A. Mattei, P. Jemth, S. Gianni

{"title":"Understanding the role of phosphorylation in the binding mechanism of a PDZ domain","authors":"A. Toto, A. Mattei, P. Jemth, S. Gianni","doi":"10.1093/protein/gzw055","DOIUrl":"https://doi.org/10.1093/protein/gzw055","url":null,"abstract":"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. The results show that phosphorylation of a serine residue in the RIL peptide has dual and opposing effects: it increases both the association and dissociation rate constants, which leads to an overall weakening of binding. Furthermore, we performed binding experiments with a RIL peptide in which the serine was replaced by a glutamate, a commonly used method to mimic phosphorylation in proteins. Strikingly, both the affinity and the ionic strength dependence of the affinity differed markedly for the phosphoserine and glutamate peptides. These results show that, in this particular case, glutamate is a poor mimic of serine phosphorylation.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79481611","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: 15

Direct determination of enzyme kinetic parameters from single reactions using a new progress curve analysis tool 用新的进展曲线分析工具直接测定单反应的酶动力学参数

Protein Engineering, Design and Selection Pub Date : 2016-10-15 DOI: 10.1093/protein/gzw053

Felix K. Bäuerle, Á. Zotter, G. Schreiber

引用次数: 14

Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation 设计一个最小的G蛋白，以促进G蛋白偶联受体在其活性构象中的结晶

Protein Engineering, Design and Selection Pub Date : 2016-09-26 DOI: 10.1093/protein/gzw049

B. Carpenter, C. Tate

{"title":"Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation","authors":"B. Carpenter, C. Tate","doi":"10.1093/protein/gzw049","DOIUrl":"https://doi.org/10.1093/protein/gzw049","url":null,"abstract":"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. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79524110","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: 105

Structural features determining thermal adaptation of esterases. 决定酯酶热适应性的结构特征

Protein Engineering, Design and Selection Pub Date : 2016-02-01 Epub Date: 2015-12-07 DOI: 10.1093/protein/gzv061

Filip Kovacic, Agathe Mandrysch, Chetan Poojari, Birgit Strodel, Karl-Erich Jaeger

{"title":"Structural features determining thermal adaptation of esterases.","authors":"Filip Kovacic, Agathe Mandrysch, Chetan Poojari, Birgit Strodel, Karl-Erich Jaeger","doi":"10.1093/protein/gzv061","DOIUrl":"10.1093/protein/gzv061","url":null,"abstract":"<p><p>The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10°C to 70°C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function. </p>","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80983072","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

Overcoming a species-specificity barrier in development of an inhibitory antibody targeting a modulator of tumor stroma 在肿瘤基质调节剂的抑制抗体开发中克服物种特异性障碍

Protein Engineering, Design and Selection Pub Date : 2016-01-26 DOI: 10.1093/protein/gzv067

I. Grossman, T. Ilani, S. Fleishman, D. Fass

{"title":"Overcoming a species-specificity barrier in development of an inhibitory antibody targeting a modulator of tumor stroma","authors":"I. Grossman, T. Ilani, S. Fleishman, D. Fass","doi":"10.1093/protein/gzv067","DOIUrl":"https://doi.org/10.1093/protein/gzv067","url":null,"abstract":"The secreted disulfide catalyst Quiescin sulfhydryl oxidase-1 (QSOX1) affects extracellular matrix organization and is overexpressed in various adenocarcinomas and associated stroma. Inhibition of extracellular human QSOX1 by a monoclonal antibody decreased tumor cell migration in a cell co-culture model and hence may have therapeutic potential. However, the species specificity of the QSOX1 monoclonal antibody has been a setback in assessing its utility as an anti-metastatic agent in vivo, a common problem in the antibody therapy industry. We therefore used structurally guided engineering to expand the antibody species specificity, improving its affinity toward mouse QSOX1 by at least four orders of magnitude. A crystal structure of the re-engineered variant, complexed with its mouse antigen, revealed that the antibody accomplishes dual-species targeting through altered contacts between its heavy and light chains, plus replacement of bulky aromatics by flexible side chains and versatile water-bridged polar interactions. In parallel, we produced a surrogate antibody targeting mouse QSOX1 that exhibits a new QSOX1 inhibition mode. This set of three QSOX1 inhibitory antibodies is compatible with various mouse models for pre-clinical trials and biotechnological applications. In this study we provide insights into structural blocks to cross-reactivity and set up guideposts for successful antibody design and re-engineering.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-01-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88120794","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: 11

Computer-aided antibody design 计算机辅助抗体设计

Protein Engineering, Design and Selection Pub Date : 2012-06-02 DOI: 10.1007/978-1-0716-2609-2

Daisuke Kuroda, H. Shirai, M. Jacobson, Haruki Nakamura

引用次数: 254