Protein Engineering, Design and Selection最新文献

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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

The Mo/Cu-dependent CO dehydrogenase from Oligotropha carboxidovorans is an enzyme that is able to catalyze CO oxidation to CO2; moreover, it can also oxidize H2, thus eliciting a characteristic EPR signal. Interestingly, the Ag-substituted enzyme form proved unable to catalyze H2 oxidation. In the present contribution, we characterized the reactivity of the enzyme with H2 by quantum-chemical calculations. It was found that dihydrogen binding to the wild-type enzyme requires significant structural rearrangements of the active site Theoretical EPR spectra for plausible H2-bound models of the partially reduced, paramagnetic active site are also presented and compared with the experimental counterpart. Finally, density functional theory modeling shows that Ag substitution impairs H2 binding at the active site.

来自低聚菌的Mo/ cu依赖的CO脱氢酶是一种能够催化CO氧化为CO2的酶;此外，它还可以氧化H2，从而引发一个特征性的EPR信号。有趣的是，ag取代的酶形式被证明不能催化H2氧化。在本贡献中，我们用量子化学计算表征了酶与H2的反应性。研究发现，二氢与野生型酶的结合需要明显的活性位点结构重排，并给出了部分还原的顺磁活性位点的h2结合模型的理论EPR谱，并与实验结果进行了比较。最后，密度泛函理论模型表明Ag取代破坏了活性位点的H2结合。

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

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.

来自谷氨棒状杆菌和其他生物的龙胆酸1,2-双加氧酶(GDOs)氧化裂解龙胆酸(2,5-二羟基苯甲酸酯)的芳香核，但不能转化水杨酸(2-羟基苯甲酸酯)。相反，变形杆菌水杨酸假氨基杆菌合成一种酶(“水杨酸双加氧酶”，SDO)，它可以裂解龙胆酸，但也可以(取代)水杨酸。序列比较表明，该GDO与C. glutamum ATCC 13032的GDO同属于主要来源于革兰氏阳性菌的GDO群。将序列比较与先前对SDO进行的结构和突变分析相结合，可以确定一个氨基酸残基(Ala112)，该残基可能阻止谷氨酸丙氨酸GDO氧化(取代)水杨酸盐。因此，将相关突变(Ala→Gly)引入谷氨酸丙氨酸GDO中。获得的GDO变体获得了氧化水杨酸酯和其他几种单羟基化底物的能力。为了筛选更广泛的酶变异，一种显色试验被开发出来，允许检测转化水杨酸的细菌菌落。通过对不同位置饱和诱变获得的一组GDO变异进行筛选，验证了该检测系统的适用性。这表明携带突变Ala112→Ser、Ala112→Ile和Ala112→Asp的GDO变体也转化为水杨酸盐。

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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

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.

设计锚蛋白重复序列蛋白(DARPin®)是一种人工非免疫球蛋白结合蛋白，具有潜在的治疗分子应用前景。DARPin 6G9以高亲和力结合白细胞介素-13并阻断信号通路，因此有望用于治疗哮喘和其他特应性疾病。在高分辨率下测定了DARPin 6G9的非结合形态和与IL-13配合物的晶体结构。DARPin与IL-13受体链13R&agr 1竞争相同的表位，但不干扰另一个受体链IL-4R&agr的结合。对晶体中多个拷贝的DARPin分子的分析表明，分子动力学模拟预测了n端帽的构象不稳定性。将游离状态和与IL-13复合物的DARPin结构进行比较，发现在结合时锚蛋白重复序列的协同运动导致了结合位点的打开。DARPin 6G9采用的诱导配合模式对DARPin来说是非常不寻常的，因为它们被设计为特别稳定和刚性的分子。这一发现表明，DARPins可以通过各种结合机制起作用，并表明支架中的一些灵活性可能是一种优势。

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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

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.

PDZ结构域是哺乳动物中最常见的蛋白-蛋白相互作用结构域之一。虽然一些研究已经证明了磷酸化在涉及PDZ结构域的相互作用中的重要性，但缺乏详细的机制数据来解决磷酸化如何影响PDZ相互作用。在这里，我们通过平衡和动力学结合实验解决了这个问题，利用蛋白酪氨酸磷酸酶L1的PDZ2及其与天然配体RIL的肽的相互作用。结果表明，RIL肽中丝氨酸残基的磷酸化具有双重和相反的作用:它增加了结合和解离速率常数，从而导致结合的整体减弱。此外，我们对RIL肽进行了结合实验，其中丝氨酸被谷氨酸取代，这是一种常用的模拟蛋白质磷酸化的方法。引人注目的是，无论是亲和力和离子强度依赖的亲和力的磷酸丝氨酸和谷氨酸肽明显不同。这些结果表明，在这种特殊情况下，谷氨酸是一种很差的丝氨酸磷酸化模拟物。

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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

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. The third model is a direct simulation of the enzyme kinetics. Depending on the chosen model, the tools excel in speed, accuracy or initial value requirements. Using simulated and experimental data, we show the strengths and pitfalls of the different fitting models. Direct simulation proves to have the highest level of accuracy, but it also requires reasonable initial values to converge. Finally, we propose a standard procedure to obtain optimized enzyme kinetic parameters from single progress curves.

随着基于计算机的数据拟合方法成为生物化学的标准工具，酶动力学的进程曲线分析是一种可行但很少使用的工具。在这里，我们提出了一个多功能的基于matlab的工具(PCAT)来分析催化过程曲线与三种互补的模型方法。前两个模型基于该问题已知的封闭形式解:第一个模型用解析近似描述所需的Lambert W函数，第二个模型提供Lambert W函数的数值解。第三种模型是对酶动力学的直接模拟。根据所选择的模型，这些工具在速度、精度或初始值要求方面表现出色。通过模拟和实验数据，我们展示了不同拟合模型的优点和缺陷。直接模拟被证明具有最高的精度，但它也需要合理的初始值来收敛。最后，我们提出了从单个过程曲线获得优化酶动力学参数的标准程序。

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

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.

G蛋白偶联受体(gpcr)在响应细胞外刺激时调节细胞质信号，是多种疾病的重要治疗靶点。确定所有激活状态下gpcr的结构对阐明信号转导的精确机制和促进优化药物设计具有重要意义。然而，由于其固有的不稳定性，gpcr与细胞质信号蛋白(如异源三聚体G蛋白和β-阻滞蛋白)复合物的结晶被证明是具有挑战性的。在这里，我们描述了一种最小G蛋白的设计，mini-Gs，它仅由腺苷酸环化酶刺激G蛋白Gs的GTPase结构域组成。Mini-Gs是一种小的可溶性蛋白，在没有Gβγ亚基的情况下有效地偶联gpcr。我们利用合理设计的诱变技术改造了mini-Gs，使其与洗涤剂溶解的β1-肾上腺素能受体(β1AR)形成稳定的复合物。迷你G蛋白在gpcr中诱导类似于异三聚体G蛋白的药理学和结构变化，但消除了与这些复合物结晶相关的许多问题，特别是它们的大尺寸、构象动力学和洗涤剂中的不稳定性。因此，它们是一种新颖的工具，可以促进gpcr活性构象的生化和结构表征。

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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

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.

微生物要适应极端的生活温度，就必须进化出在这些条件下具有高催化效率的酶。这种嗜极酶是研究蛋白质稳定性、动力学和功能之间关系的宝贵工具。尽管如此，人们对温度对酶的结构和功能的多重影响在分子水平上仍然知之甚少。我们对从生活在 10°C 至 70°C 温度范围内的细菌中分离出来的四种同源酯酶进行了分析，发现了通过优化蛋白质表面的局部柔韧性来调节蛋白质热特性的适应途径。虽然重组酯酶的生化特性保持不变，但它们的热特性已经进化到类似于各自细菌栖息地的特性。在酶催化的最佳温度附近进行的分子动力学模拟显示，四个表面暴露环的灵活性与温度有关。一些环路的灵活性随着温度的升高而增加，随着温度的降低而降低，这与那些有助于蛋白质稳定性的环路所预期的一样。这些区域保持灵活性似乎对酶的正常功能很重要。远离活性位点的这四个环的结构差异远远大于整个蛋白质结构的差异，这表明这些环内的氨基酸交换更为频繁，从而使细菌能够根据不同的温度要求调整原子间的相互作用，而不影响酶的整体功能。

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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

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.

分泌的二硫催化剂Quiescin巯基氧化酶-1 (QSOX1)影响细胞外基质组织，并在各种腺癌和相关基质中过表达。在细胞共培养模型中，单克隆抗体抑制细胞外的人QSOX1可减少肿瘤细胞的迁移，因此可能具有治疗潜力。然而，QSOX1单克隆抗体的物种特异性一直是评估其作为体内抗转移剂效用的一个挫折，这是抗体治疗行业的一个常见问题。因此，我们使用结构引导工程来扩大抗体的物种特异性，将其对小鼠QSOX1的亲和力提高了至少四个数量级。重组变体的晶体结构与小鼠抗原的复合物表明，该抗体通过改变其重链和轻链之间的接触，以及用灵活的侧链和多功能的水桥极性相互作用取代庞大的芳烃来实现双物种靶向。同时，我们制作了一种针对小鼠QSOX1的替代抗体，该抗体显示出新的QSOX1抑制模式。这组三种QSOX1抑制抗体与多种小鼠模型兼容，可用于临床前试验和生物技术应用。在这项研究中，我们提供了对交叉反应的结构块的见解，并为成功的抗体设计和重组建立了指南。

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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

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